High efficiency and high precision microfluidic devices and methods

ABSTRACT

New high density microfluidic devices and methods provide precise metering of fluid volumes and efficient mixing of the metered volumes. A first solution is introduced into a segment of a flow channel in fluidic communication with a reaction chamber. A second solution is flowed through the segment so that the first solution is displaced into the reaction chamber, and a volume of the second solution enters the chamber. The chamber can then be isolated and reactions within the chamber can be initiated and/or detected. High throughput methods of genetic analysis can be carried out with greater accuracy than previously available.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/881,627 filed Jan. 19, 2007, and U.S. Provisional Application No.60/934,499 filed Jun. 13, 2007, the contents of each which isincorporated herein by reference.

BACKGROUND

High density microfluidic devices are useful in a wide range ofresearch, diagnostic and synthetic applications, including immunoassays,nucleic acid amplification and genomic analysis, cell separation andmanipulation, and synthesis of radionuclides, organic molecules, andbiomolecules. The advantages of microfluidic devices includeconservation of reagents and samples, high density and throughput ofsample analysis or synthesis, fluidic precision and accuracy, and aspace reduction accompanying the replacement of counterpart equipmentoperating at the macrofluidic scale.

However, the manipulation of fluid volumes on the order of nanolitersand picoliters has required many new discoveries and design innovations.There are fundamental differences between the physical properties offluids moving in large channels and those traveling throughmicrometer-scale channels. See, e.g., Squires and Quake, 2005, Rev. Mod.Phys. 77, 977-1026; Stone et al., 2004, Annu. Rev. Fluid Mech.36:381-411; and Beebe et al., 2002, Ann. Rev. Biomed. Eng. 4:261-86. Forexample, at a microfluidic scale the Reynolds number is extremely small,reflecting a difference in the ratio of inertial to viscous forcescompared to fluids at macroscale. Fluids flowing in microfluidic systemsexhibit reduced turbulence, electro-osmotic and laminar flow properties,and in other ways behave differently than observed at a macroscale.There remains a need for new approaches to effecting efficient flow,containment and mixing of microfluids.

BRIEF SUMMARY OF THE INVENTION

New high density microfluidic devices and methods provide precisemetering of fluid volumes and efficient mixing of the metered volumes. Afirst solution is introduced into a segment of a flow channel in fluidiccommunication with a reaction chamber. A second solution is flowedthrough the segment so that the first solution is displaced into thereaction chamber, and a volume of the second solution enters thechamber. The chamber can then be isolated and reactions within thechamber can be initiated and/or detected. High throughput methods ofgenetic analysis can be carried out with greater accuracy thanpreviously available.

In one aspect the invention provides a microfluidic device comprising anarray of fluidically interconnected unit cells, where each unit cellcomprises (a) a first microfluidic flow path in fluid communicationwith: 1) a reaction chamber and 2) a first microfluidic bus line, wherethe first microfluidic bus line is in fluidic communication with asample source reservoir; b) a first valve situated in the firstmicrofluidic flow path; c) a second valve situated in the firstmicrofluidic flow path; d) a second microfluidic flow path in fluidiccommunication with: 1) the first microfluidic flow path at a junctionbetween the first and second valves, and 2) a second microfluidic busline, where the second microfluidic bus line is in fluidic communicationwith a reagent source reservoir e) a third valve situated 1) in thesecond microfluidic flow path, or 2) in the second microfluidic bus linepositioned between the microfluidic flow channel of the unit cell andthe second microfluidic flow path of an adjacent unit cell.

In one aspect the invention provides a microfluidic device comprising anarray of fluidically connected unit cells, where each unit cellcomprises: a) a reaction chamber; b) a first microfluidic flow pathhaving a proximal end and a distal end, where the first microfluidicflow path (i) is in fluidic communication at its proximal end with thereaction chamber and (ii) is in fluidic communication at its distal endwith a first microfluidic bus line, where the first microfluidic busline is in fluidic communication with (i) a sample source reservoir and(ii) a plurality of unit cells; c) a first valve and a second valve,where (i) the second valve is situated in the first microfluidic flowpath and (ii) the first valve situated in the first microfluidic flowpath between the first valve and the reaction chamber; where the fluidcapacity of the segment of the first microfluidic flow path between thefirst valve and the second valve is less than the fluid capacity of thereaction chamber; d) a second microfluidic flow path in fluidiccommunication with the first microfluidic flow path at a junctionlocated between the first and second valves; and in fluidiccommunication with a second microfluidic bus line, where the secondmicrofluidic bus line is in fluidic communication with (i) a reagentsource reservoir and (ii) a plurality of unit cells; e) a third valvesituated a second microfluidic flow path between the second microfluidicbus line and the junction or in the second microfluidic bus linepositioned between the microfluidic flow channel of the unit cell andthe second microfluidic flow path of an adjacent unit cell; where thearray comprises at least 16 unit cells grouped into at least 4 firstsets (e.g., rows) and at least 4 second sets (e.g., columns) of unitcells; where the at least 16 unit cells are not members of more than onefirst set or more than one second set; where each unit cell of each thefirst set is fluidically linked to a different first microfluidic busline; and where each unit cell of each the first set is fluidicallylinked to a different second microfluidic bus line.

In some versions of the device the third valve is situated in the secondmicrofluidic flow path. In some versions of the device the reactionchamber is a dead-end chamber. In some versions, the array has at least1000 unit cells. In some versions of the device, there is a check valvein the first microfluidic flow path between the reaction chamber and thefirst valve. In some versions of the device the second valve and/or thethird valve is a check valve permitting flow only toward the reactionchamber.

The device is fabricated from elastomeric material(s). In some versions,the device has channel segments in at least two different levels of thedevice. For example, the first microfluidic flow path may have channelsegments in at least two different levels of the device. In someversions, the device includes at least one fluid communication via. Insome versions of the device the first and second valves are regulated bya single push-up/push-down valve. In some versions the device may have avalve system with i) three on-off valves or ii) three on-off valves andone check valve or iii) two on-off and one or two check valves or iv)one on-off valve and two or three check valves.

In one aspect a method for mixing solutions in a microfluidic device isprovided. The method may include the steps of introducing a firstsolution into a segment of a flow channel, where the flow channel is influidic communication with a reaction chamber; flowing a second solutionthrough the segment, thereby displacing the first solution into thereaction chamber; and flowing the second solution into the reactionchamber, whereby mixing of the first and second solutions occurs in thereaction chamber. The method may include the further step of fluidicallyisolating the reaction chamber.

In embodiment the method may include i) introducing a first solutioninto a segment of a flow channel of a unit cell of the device, where theflow channel is in fluidic communication at a first end with a reactionchamber having fluid capacity N; where a first position in the flowchannel is proximal to a second position if the first position isfluidically closer to the reaction chamber the second position, and isdistal to the second position if the first position is fluidicallyfurther from the reaction chamber than the second position; where theflow channel segment is bounded by a first valve located in the flowchannel and a second valve located in the flow channel, where the secondvalve is distal to the first valve; where the flow channel segment is influidic communication, through a first input junction, with a source ofthe first solution, the first input junction being positioned betweenthe first valve and the second valve; where the flow channel segment isin fluidic communication, through a second input junction, with a sourceof the second solution, the second input junction being positioneddistal to the second valve, where flow of the second solution from thesource to is regulated by a third valve; where the fluid capacity of theflow channel segment is less than N; and where the first valve is closedwhen the first solution is introduced; where the second valve is in theclosed when the first solution is introduced, or is a check valve thatpermits flow only toward the reaction chamber; where the third valve ispositioned such that when the first valve is closed, the second valve isclosed or is a check valve, and the third valve is closed or is a checkvalve that permits flow only toward the reaction chamber, the firstsolution is retained in the segment. If the third valve is not acheck-valve, the method includes the step of closing the third valve.The method also includes the steps of iii) introducing the secondsolution into the segment. If the second valve is not a check valve, theintroducing includes the steps of 1) flowing the second solution into aportion of the flow channel distal to the second valve before, after orconcurrently with step (i) 2) opening the second valve; 3) opening thefirst valve; 4) flowing the second solution into the segment of the flowchannel, thereby displacing the first solution into the reactionchamber; and 5) flowing the second solution into the reaction chamber.If the second valve is a check valve the introducing includes the stepsof 1) flowing the second solution into the portion of the flow channeldistal to the second valve; 2) opening the first valve; 3) flowing thesecond solution through the second valve, thereby displacing the firstsolution into the reaction chamber, and 4) flowing the second solutioninto the reaction chamber; whereby the first solution and the secondsolution are mixed in the reaction chamber. The method may include thefurther step of closing the first valve.

In some embodiments the device comprises an array of at least 1000, andthe unit cells are grouped in an array comprising M rows of unit cellsand N columns of unit cells, M>25 and N>25. In some embodiments the samefirst solution is introduced into each unit cell of a column and thesame second solution is introduced into each unit cell of a row. In someembodiments first solutions contain reagents for amplification of anucleic acid and the second solutions contain a nucleic acid sample.

In a related aspect the invention provides a method for combining two ormore solutions in a microfluidic reaction chamber by introducing apredetermined volume of a first solution into a reaction chamber,introducing a predetermined volume of a second solution into a reactionchamber, and fluidically isolating the reaction chamber. Preferably thecombined volumes of the first solution and the second solution equalsthe fluidic capacity of the reaction chamber. Preferably the volume ofone of the solutions is at least twice the volume of the other solution.

BRIEF DESCRIPTION OF THE FIGURES

The figures are to illustrate feature of the invention and are not drawnto scale.

FIG. 1 is an illustration of flow paths and valves of a FCS device(Exemplar 1) showing a 4×4 array of cells. The cells are grouped bycolumn (C1, C2, C3, C4) and row (R1, R2, R3, R4).

FIG. 2 is an illustration of a single cell of the Exemplar 1 FCS deviceshown in FIG. 1.

FIGS. 3A-3H illustrate filling and use of the Exemplar 1 FCS device.

FIG. 4 is an illustration of flow paths and valves of a FCS device(Exemplar 2) showing a 2×2 array of cells.

FIG. 5 is an illustration of an individual cell of a FCS device(Exemplar 3).

FIG. 6 is an illustration of flow paths and valves of an individual cellof an FCS device (Exemplar 4).

FIG. 7A-C illustrates injection of multiple reagent slugs in an FCSdevice (Exemplar 5).

FIG. 8 shows a push-up/push down valve.

FIGS. 9A and 9B illustrate an FCS device including a check valve. FIG.9C illustrates a FCS device in which the chamber 400 has a valved exitchannel 500.

FIG. 10 shows a microfluidic check valve.

FIG. 11 illustrates a method for fabrication of a microfluidic checkvalve.

DETAILED DESCRIPTION

Rapid and efficient mixing in microfluidic devices is often critical tothe effective function of a microfluidic device. However, the mechanicsof mixing between two separated liquids in the microfluidic channels isquite different from the macro world. For a micrometer size channel, thetypical value of diffusion coefficients, D, range between 10⁻⁵ cm²s⁻¹ atthe high end (small molecule) to 10⁻⁷ cm²s⁻¹ at the low end (largemolecule, such as genomic DNA). The diffusion time scale, τ˜L²/D, (L isthe length of character channel) may be seconds for a small molecule ina channel of 10 μm or hours for a large molecule in a channel of 100 μm.This invention relates to microfluidic devices and methods of using thedevices that provide precise metering of fluid volumes and efficientmixing of the metered volumes. The methods, devices and componentsdisclosed herein are useful for analytical assays for research ordiagnostic purposes where high density, high throughput, sampleparsimony, and lower cost are desired. The devices and methods are alsouseful as tools for the synthesis, sorting, and storage of high valuechemical and biological entities, and other uses that will be apparentto the skilled reader.

I. OVERVIEW

The microfluidic devices of the present invention utilize aconfiguration in which at least one solution is metered into a segmentof a flow channel, typically through a junction disposed between valvesalong the flow channel. The junction typically has an on/off valve or aone way check valve at the inlet portion of the junction. The flowchannel valves that bracket the junction are closed and the junctioninlet valve is opened. A solution is instilled into the segment of theflow channel. The filling of the segment is preferably performed by“blind filling” the segment. Blind filling takes advantage of thepermeability of the material defining at least one side of the flowchannel to gases and not to liquid. The first solution is filled intothe flow channel segment by placing the solution under pressure at thejunction and allowing the first solution to fill the segment as thegases that are present in the flow channel diffuse out through the gaspermeable material. Once the segment of the flow channel defined by thevalves is filled, the inlet junction is closed or allowed to close and aprecisely defined volume is contained within the flow channel segment.The exact volume is determined by the flow channel dimensions and thespacing of the valves along the segment that are closed to define theblind filled portion of the flow channel. With the flow channel valvesremaining closed, a second solution is introduced into an empty portionof the flow channel by blind filling against one of the closed valves.In one embodiment, the empty portion of the flow channel is adjacent tothe segment of the flow channel. By maintaining the second solutionunder pressure and then opening the flow channel valves on the flowchannel segment, the second solution pushes the first solution throughthe flow channel. In a preferred embodiment, the flow channel segmentvalve opposite the valve against which the second solution is blindfilled, is adjacent to a reaction chamber of a defined volume. When theflow channel segment valves are opened, the second solution pushes thefirst solution into the reaction chamber. By fabricating the reactionchamber such that the reaction chamber volume is greater that the volumeof the first solution in the flow channel, a precisely defined amount ofthe second solution is pushed into the reaction chamber along with theknown volume of the first solution. The volume of the second solutionthat fills the reaction chamber is defined by the volume of the reactionchamber minus the volume of the first solution. As both solutions fillthe reaction chamber, mixing of the solutions occurs.

II. DEFINITIONS

The following definitions are provided to assist the reader. In somecases, terms with commonly understood meanings in the microfluidic artsare defined herein for clarity and/or for ready reference, and theinclusion of such definitions herein should not be construed torepresent a substantial difference over the definition of the term asgenerally understood in the art.

As used herein, “mixing” has its usual meaning. Two (or more) differentsolutions (e.g., aqueous solutions) are completely mixed when they arecombined to produce a single homogenous solution. Put differently, afirst solution containing a first solute and a second solutioncontaining a second solute produce, when completely mixed, a solution inwhich both solutes are homogenously distributed. On a microfluidic (lowReynolds number) scale, mixing is almost exclusively diffusional ratherthan turbulent. Without intending to be bound by a specific mechanism,the present invention provides superior mixing by increasing the contactarea (interface) between the solutions relative to prior microfluidicmethods of combining solutions. Using methods of the invention, a largerinterface between solutions is achieved both in the slug channel andreaction chamber. By increasing the surface area, the rate ofdiffusional mixing is increased.

As used herein, “flow channel” means a microfluidic flow channel. Amicrofluidic flow channel is a tube through which a solution (e.g., anaqueous solution) can flow. The flow channel may have a circular,rectangular or other shape cross section(s), and may have differingcross-sections or dimensions along its length. A microfluidic flowchannel is characterized by cross-sectional dimensions less than 1000microns. Usually at least one, and preferably all, cross-sectionaldimensions are less than 500 microns. Frequently at least one, andpreferably all, cross-sectional dimensions are less than 250 microns.Other exemplary flow channel dimensions are discussed herein below (see,e.g., Section VII).

As used herein, a “segment” of a flow channel refers to a section or aspecified region of a flow channel. Usually the segment is bounded byspecific structural elements of the flow channel, and thus can bedefined by reference to the structural elements. Examples of structuralelements include valves, changes in channel shape or dimensions (forexample a change from a rectangular cross-section to a circular crosssection, as when moving from a horizontal channel segment into avertical fluid communication via), change in direction (for example a“L”-shaped flow channel can be described as having two orthogonallyoriented flow channel segments), junctions with other channels,junctions with other elements (e.g., reaction chamber) and the like.Specified flow channel segments can overlap. For example, in a flowchannel with four valves designated a, b, c and d, flow channel segmentscan include a-b, a-c, a-d, b-c, b-d, and c-d. It will be apparent that aflow channel can also be referred to as a channel segment, bounded bythe termini of the channel.

As used herein, “linking segment” refers a channel segment that linkschannel segments in different layers of a device or links a channelsegment in one layer to a reaction chamber in a different layer(s). A“fluid communication via” is an example of a linking segment and refersto flow channel segment in an multilayer device that connects fluidicelements in different layers of the device and which is fabricated bydrilling, ablating (laser punching), molding or embossing a tunnelthrough the material from which the device is constructed. Anotherexample of a linking segment is a connecting channel created using areplica molding process such as that described in Anderson et al., U.S.Pat. No. 6,645,432.

As used herein, a “flow path” describes a channel segment or series ofchannel segments through which a solution can flow and, morespecifically, through which solution flows during the operation of adevice. For example, during operation of the device illustrated in FIG.6 a first solution flows from valve V2, through channel segments 250 a,b and c, and into reaction chamber 400. This path can be described as aflow path and, in this example, can be defined as the shortest fluidicpath (i.e., shortest path through which a solution can flow) from V2 toreaction chamber 400, passing through valve V1. As discussed below,during operation of the device illustrated in FIG. 6, a second solutionflows from channel 230 to reaction chamber 400. This second flow pathcan be described as the shortest path from channel 230 to reactionchamber 400. This flow path could also be described as the shortest pathfrom channel 230 to reaction chamber 400, passing through valve V3.

As used herein, the terms “layer” and “level” have the standard meaningin the art. The terms are used interchangeably when referring to theposition of flow channel segments, control channels, reaction chambersand other elements of a microfluidic device. In some microfluidicdevices channels are located in different planes of the device. Forexample, an on/off elastomeric valve can be fabricated by locating acontrol channel in one plane so that it crosses the path of a flowchannel in an adjacent different plane (see, e.g., Section VII, infra).The term “layer” also reflects the method of fabrication of suchdevices, in which layers of elastomeric structures may be bonded to eachother.

As noted above, the term “blind filling” refers to the process ofinstilling a solution into a channel or chamber that does not have afunctional exit through which an aqueous solution can flow. A chamber orchannel may have no functional exit because all potential exit flowchannels are blocked by closed or impassable valves, or because thereare no exit flow channels (e.g., no channels contiguous with the chamberother then the flow channel though which solution enters the chamber).In the latter situation, a reaction chamber into which the solution isinstilled can be called a “dead-end” reaction chamber. A flow channel,or flow channel segment, into which solution is being instilled can becalled a “dead-end” or “blind” channel. Blind filling takes advantage ofthe permeability of the material (e.g., elastomeric materials) definingat least a portion (e.g., at least a portion of one side) of the flowchannel or at least a portion (e.g., at least a portion of one wall) ofa chamber to gas and not to liquid.

As used herein, the term “check valve” refers to a one-way valve thatprevents reverse flow through a microfluidic channel. Exemplary checkvalves are described below in Example 6. However, check valves used inthe devices of the invention are not limited to the particular designillustrated in Example 6.

As used herein, a “bus line” (e.g., reagent bus line or sample bus line)refers to a flow channel or flow path in fluidic communication with asource reservoir (e.g., reagent source reservoir or sample sourcereservoir) and with slug channels or multiple unit cells. The sample busline is arranged so that a sample solution can flow from a sample sourcereservoir to slug channels without flowing though reagent bus lines orreagent input lines. The reagent bus line is arranged, if present, sothat a reagent solution can flow from a reagent source reservoir to slugchannels without flowing though sample bus lines.

Several terms, examples of which follow, are used for convenience in thediscussion and have meaning relative to each other.

The terms “vertical” and “horizontal” are used herein to describe therelationships of device elements, such as channels, and have meaningrelative to each other. It is often convenient to fabricate amicrofluidic device that is cuboid with one dimension being considerablyshorter than the other two dimensions and operate the device so that theshort dimension (height) is vertically oriented relative to the earthand the other two dimensions (length and width) are horizontallyoriented. In such a design a channel segment in which solution flows inthe height dimension may be termed “vertical” and a channel segment inwhich solution flows in the width and/or length dimension may be termed“horizontal.” However the use of these terms does not require acuboid-shaped device or operation in such an orientation.

The terms “sample solution” and “reagent solution” are used throughoutthe description to refer to solutions that are mixed using the methodsand devices of the invention. Typically a sample solution containsbiological material from a particular source (e.g., human, animal, lake,food, etc.) and a reagent solution contains compound used for analysisof a property of the sample. See Section VIII, infra, for examples.However, these terms are used for convenience and the invention is notlimited to a narrow interpretation of a “sample” and a “reagent.” Theinvention provides for methods and devices for the thorough mixing oftwo solutions. Thus, the term sample solution(s) could interchanged with“first solution(s),” “reagent solutions(s),” “analyte solutions,”“second solution(s),” etc., and the term reagent solution(s) couldinterchanged with “first solution(s),” “sample solutions(s),” “analytesolutions,” “second solution(s),” etc. For example, a first solutioncould contain one reactant and the second solution could contain adifferent reactant that when mixed chemically combine to produce areaction product.

As used herein, the terms “column” and “row” have their usual meaningsand are used in descriptions of unit cell arrays. However, no furtherfunction or structure is intended by such references. For example,reference to reagent bus lines that link columns of unit cells andsample bus lines that link rows of unit cells would be equivalent to areference to reagent bus lines that link rows of unit cells and samplebus lines that link columns of unit cells. Moreover, unless otherwisespecified, rows and columns do not require strict alignment. Unit cellsin a row, for example, can be staggered or offset from a central linerelative to each other. Further, the term “array” is not limited toarrangements of rows and column. For example, unit cells in a unit cellarray could be arranged in concentric circles, along radii of theoutermost circle.

III. FCS DEVICES

The invention relates to microfluidic assays and reactions as well asmicrofluidic devices useful for carrying out the assays and reactions.As noted above, these methods allow precise metering of fluid volumes(and therefore of quantities of assay components) and efficient mixingof the metered volumes. For convenience, devices of the invention may bereferred to as “FCS devices.”

In one preferred embodiment the FCS device is constructed fromelastomeric material(s) using multilayer soft lithography and can bereferred to as “elastomeric devices.” In this section generalfamiliarity with construction and use of elastomeric microfluidicdevices will be assumed. Additional described guidance is provided belowin Section VII and references cited therein, as well as other scientificand patent publications readily available to the ordinarily skilledartisan. Elastomeric devices have several advantages over devices madeusing other technologies. One advantage is the availability ofintegrated elastomeric valves to regulate movement of solutions.Integrated elastomeric valves are characterized by an elastomericmembrane that may be deflected into (or out of) a flow channel to blockor permit movement of solutions through the channel. A second advantageis the ability when using an elastomeric device to use blind filling toload a chamber, channel or channel segment. Notwithstanding theseadvantages, the methods of the present invention may be carried outusing other types of microfluidic devices, including hybrid devices(e.g., comprising elastomeric valves and vents, and nonelastomericmaterials to define flow paths and/or chambers), devices usingnonelastomeric valves (e.g., valves fabricated using thermoresponsivepolymer gels), and devices fabricated wholly from nonelastomericmaterials. For convenience, the description below is framed primarilywith reference to devices constructed from elastomeric material usingmultilayer soft lithography.

Reference to the figures will aid in understanding the invention.

FIG. 1 illustrates certain general features of a FCS microfluidicdevice. Other illustrative architectures are shown in FIGS. 4, 5, 6, 7and 9. As will become clear from the discussion below, the FCSmicrofluidic devices are not limited to the specific architectures shownin the figures.

FCS devices comprise an array of interconnected “unit cells” or “cells,”which form a basic unit of the device. For illustration, FIG. 1 shows a4×4 array of such cells, and FIG. 2 illustrates an individual cell.

The FCS device cell is characterized by the following features:

1. A “reaction chamber” (400). The reaction chamber may have a varietyof shapes (cubical, cylindrical, etc.). Typically the chamber has avolume in the range 1 nL to 1 uL, more often in the range 4 nL to 200nL. Usually at least one dimension is at least 50 um, and usually atleast 100 um. Particular features of reaction chambers are discussedbelow in Section VII.

2. A “slug channel” (250). A slug channel is a flow path in fluidiccommunication with the reaction chamber and with a “sample sourcereservoir” (discussed below). A slug channel may be a straight or curvedchannel in a single level of the device (see, e.g., FIG. 2), or maycomprise two or more straight or curved channel segments in differentlevels of the device connected by one or more linking segments such as afluid communication via (see, e.g., FIG. 6). The slug channel comprisesthe shortest path from valve V1 to valve V2 (discussed immediatelybelow). It is sometimes useful to refer to the “slug path” which is aterm used to encompass the slug channel along with any fluidcommunication vias (if present) linking the slug channel to the reactionchamber or linking the slug channel to the sample bus line 220, asdiscussed below. The slug path can be described as the shortest flowpath from the sample bus line to the reaction chamber, passing throughvalve V1 and valve V2.

In some embodiments, the slug channel or slug path is the only fluidicchannel connected to the reaction chamber (e.g., solutions can enter thereaction chamber only through the slug path). That is, the reactionchamber is a dead-end reaction chamber.

3. A “first valve” (V1) situated at the proximal end of the slug channelthat, when closed, fluidically isolates the reaction chamber (400) fromthe more distal part of the slug channel. As used in this context, theterm “proximal” refers to a position in the slug path relative to thereaction chamber. An element located in the slug path at a position thatis closer to the reaction chamber than the position of a second elementis proximal relative to the second element. The second element is distalrelative to the first element.

In some embodiments, the slug path is free of valves in the segmentbetween the first valve (V1) and the reaction chamber. See, e.g., FIGS.1-7. In some embodiments a check-valve (VCK) is situated between thefirst valve (V1) and the reaction chamber to prevent reverse flow fromthe reaction chamber into the slug channel (see, e.g., FIG. 9A).

4. A “second valve” (V2) in the slug channel distal to first valve (V1).In some embodiments, the slug path is free of valves in the segmentbetween the first valve (V1) and the second valve (V2). See, e.g., FIGS.1-7. In some embodiments a check-valve (VCK) is situated between thefirst valve (V1) and the second valve (V2) (see FIG. 9B) to preventreverse flow from the reaction chamber into the slug channel. It ispreferable in such a design to place the check valve close to valve V1,to minimize the volume of sample solution in the slug path that is influidic communication with the contents of the reaction chamber afterthe reaction chamber is filled.

In general the first and second valves (V1 and V2) are controlled by thesame actuation system and are opened or closed at the same time. Forexample, in an elastomeric device as illustrated in FIGS. 1-7, valves V1and V2 are both controlled by control channel 1 (260). In alternativeembodiments, however, the second valve (V2) can be a check valve thatprevents flow of solution in the fluidic direction opposite the reactionchamber. That is, solution can flow through valve V2 towards thereaction chamber, but not in the opposite direction.

6. A “sample bus line” (220). The slug channel (250) is in fluidiccommunication with a sample bus line (220) at a junction distal to thesecond valve (V2). A sample bus line is a flow channel in fluidiccommunication with a sample source reservoir and with slug paths of aplurality of unit cells (e.g., a row of unit cells). Usually theplurality comprises at least 10 unit cells, often at least 30 unitcells, often at least 40 unit cells, and sometimes at least 96 unitcells. In some embodiments the plurality is exactly 32, 48, or 96 unitcells. Each unit cell is in fluidic communication with a single samplebus line. In some embodiments, unit cells of each row in an array arefluidically connected to a different sample bus line. Thus, in someembodiments the sample bus line constitutes a fill source for the slugpaths of a particular row. Using this arrangement the slug path of cellsof each row will be loaded with the same sample.

The sample bus line (220) may be connected to the slug channel distal tothe second valve by a fluid communication via (240) (see, e.g., FIG. 4)or other linking segment and/or by a “sample input line” (290) (see,e.g., FIG. 6). The sample input line 290 may be short (see, e.g., FIG.4).

As will be apparent, closure of the second valve (V2) prevents flow fromthe sample bus line (or sample input line) to the reaction chamber.

7. A “reagent bus line” (230). In certain embodiments the slug channelis in fluidic communication with a reagent bus line. A reagent bus lineis a bus line in fluidic communication with a reagent source and withslug channels of a plurality of unit cells (e.g., a column of unitcells). Usually the plurality comprises at least 10 unit cells, often atleast 30 unit cells, often at least 40 unit cells, and sometimes atleast 96 unit cells. In some embodiments the plurality is exactly 32,48, or 96 unit cells. Each unit cell is in fluidic communication with asingle reagent bus line. In some embodiments, unit cells of each columnin an array are fluidically connected to a different reagent bus line.

5. A “reagent input channel” (300). The reagent input channel is influidic communication with the slug channel at a junction (J1) that liesbetween the first valve (V1) and second valve (V2) (i.e., is distal tovalve V1 and proximal to valve V2). The reagent input channel is influidic communication with a reagent source reservoir. With valves V1and V2 closed, reagent solution can flow from the reagent sourcereservoir into the slug channel, filling the portion of the slug channelbetween valves V1 and V2 with solution.

In some embodiments the reagent input channel is linked to the reagentsource reservoir though a reagent bus line (230). See, e.g., FIGS. 4-6.In some embodiments the reagent input channel comprises or consists of afluid communication via, or other linking segment through which reagentsolution flows from the reagent bus line.

In some embodiments, the slug channel is not fluidically connected inthe segment between the first valve (V1) and second valve (V2) to anyinput lines other then the reagent input channel. That is, junction J1is the only junction in this segment (see, e.g., FIGS. 4-7).

In a different embodiment, as illustrated in FIG. 1, a distinct reagentbus line is not used. Instead, a reagent input channel (300) of eachcell is linked to the slug channel of an adjacent cell in the slugchannel segment bounded by the first and second valves (V1 and V2). Whenvalves V1 and V2 are closed reagent introduced into one reagent inputchannel flows to all reagent input channels in a column. In some suchembodiments, exactly two reagent input channels (one corresponding tothe cell and one corresponding to an adjacent cell) are the onlychannels in fluidic communication with the slug path in the region ofthe slug path lying between valves V1 and V2.

It will be clear that other arrangements and architectures, with orwithout bus lines may be used, so long as a reagent solution from asingle reagent source can be delivered to slug channels of a pluralityof unit cells in the slug channel segments that lie between valve V1 andvalve V2.

7. Each unit cell also comprises a “third valve” (V3) that regulatesflow from the reagent input channel to the slug channel of each cell ina column. The position of the third valve will depend on the nature ofthe reagent input channels and reagent bus line (if present). The thirdvalves may be located in each reagent input line (see FIGS. 1, 5 and 6).Alternatively the third valves may be located in the reagent bus linebetween cells (see FIG. 7). When the third valves of a column of unitcells are closed, each unit is fluidically isolated from other cells inthe same column, but remain fluidically connected through a sample busline to other cells in the row. In this embodiment the slug channels ofa given column are therefore interconnected when valve 3 is open, butcapable of being isolated from each other upon actuation of controlchannel 2 (270), as is discussed below.

Alternatively, the third valve (V3) may be a check valve that permitsfluid flow toward the unit cell reaction chamber, but does not permitflow through the valve in the reverse direction.

In some embodiments, in an FCS device sample flowing from the sample busline to the reaction chamber passes though exactly two, no more thanthree (e.g., exactly three), or no more than four (e.g., exactly four)valves. In some embodiments, sample flowing from the sample bus line tothe reaction chamber passes though exactly one check valve, or throughexactly two check valves. In some embodiments, sample flowing from thesample bus line to the reaction chamber passes though exactly twovalves, one of which is a check valve, or exactly three valves, one ortwo of which is a check valve.

As noted above, a FCS device comprises an array of FCS cells, which maybe arranged and linked so that cells of each column can be providedreagent from a common reagent reservoir, and cells of each row can beprovided a sample from a common sample reservoir. More generally, in anFCS device array unit cells are arranged in sets. The array may haveunit cells arranged in columns and rows. The cells of a particularcolumn (or row) need not be precisely aligned and may, for example, begrouped along concentric circles and radii. Any arrangement that allowsanalysis cells to be grouped into at least 4 first sets (e.g., rows)with cells of each first set being fluidically linked by a sample busline (220), and at least 4 second sets (e.g., columns) of unit cells,with cells of each second set being fluidically linked to each other bya flow path that includes the second microfluidic flow path (reagentinput line 300) of the cell, where unit cells in the array are notmembers of more than one first set or more than one second set.

In some embodiment, the FCS device comprises unit cells in columns androws in which each column is associated with a reagent bus line (230).When valve V3 is open and valves V1 and V2 are closed, solution flowingthrough the reagent bus line is delivered to the slug path of each cellin the column. Each row is associated with a sample bus line (220).Solution flowing through the sample bus line is delivered to the slugpath of each cell in the row and when valves V1 and V2 are sample flowsfrom the sample bus line of a row, through the slug path of cells in therow, and into the reaction chambers of cells of the row. Preferably eachcolumn is associated with a unique reagent bus line and each row isassociated with a unique sample bus line.

9. Source reservoirs. Source reservoir are containers, wells, chambersand the like that can be loaded with desired sample and reagentsolutions. The FCS device may comprise reagent source reservoirs andsample source reservoirs which are part of an integrated carrier device.Alternatively, channels of the device can be fluidically connected toexternal reservoirs. Generally each sample bus line (220) is in fluidiccommunication with a sample source reservoir (which is usually a uniquereservoir) and each reagent bus line (230) is in fluidic communicationwith a reagent source reservoir (which is usually a unique reservoir).In FCS devices designed without each reagent bus lines, reagent inputchannels of each column may be fluidically connected to a reagent sourcereservoir. The source reservoirs are generally not filled with solutionsuntil they are being prepared for use. However, in some embodimentsdevices are provided in which at least some reservoirs are prefilled.

10. In a FCS device using integrated elastomeric on-off valves, eachcell also comprises a portion of at least one control channel. Typicallythe device includes a “first control channel” (260), which regulatesflow through the first valve V1 and the second valve V2, and a “secondcontrol channel” (270), which regulates flow through the third flowchannel V3. The valves are opened or closed in response to pneumaticpressure in a control channel, causing deflectable membrane portions todeflect into the flow channels to stop flow of solution through a flowchannel and fluidically separate regions of a flow channel from eachother (see Section VII below). Usually the control channels are locatedin a layer of the device that is adjacent to the layer containing theregulated flow channel. In a preferred embodiment each cell comprisesportions of two control channels, a first control channel (260)regulating valves V1 and V2, and a second control channel (270)regulating valve V3. In an alternative embodiment valves V1 and V2 canbe controlled by two different control channels. In embodiments in whichvalve V3 is a one-way check valve, it is possible to omit controlchannel 2.

In one embodiment each first control channel regulates valves V1 and V2along a row of the array, and each second control channel regulatedvalves V3 along a column of the array.

An FCS device of the invention usually has at least 16 cells arrayed inat least four rows and at least four columns of cells. Preferably an FCSdevice comprises more than 16 cells. For example FCS devices have beendesigned having a 12×8 (96 cells), 12×32 (384), 32×32 array (1024cells), 48×48 (2304 cells), 96×48 array (4608 cells) and a 96×96 array(9216 cells). In certain embodiments an FCS device may have at least 50cells, at least 100, at least 500, at least 1000 cells, at least 2000cells, at least 3000 cells, at least 4000 cells, at least 7500 cells, atleast 9000 cells or an even greater number of cells. In one embodimentthe cells are contained in a 30 mm×30 mm (900 mm²) area of thenanofluidic chip. In one embodiment the cells are contained in anapproximately 31 mm×31 mm area of the nanofluidic chip. In variousembodiments the density of cells is at least 1 per mm², at least 2 permm², at least 3 per mm², at least 4 per mm², at least 5 per mm², atleast 6 per mm², at least 7 per mm², at least 8 per mm², at least 9 permm², at least 10 per mm², or more than 10 cells per mm². In variousembodiments the density of cells is from 1-20 cells per mm², or 1-11cells per mm².

IV. OPERATION OF FCS DEVICE

In the operation of an FCS device, the slug path is filled (e.g., byblind filling) with a reagent solution. The reagent is contained in asection of the slug path bounded by valves V1 and V2. A sample solutionis introduced through the sample bus line (and optionally through asample input channel), typically by blind filling, into the section ofthe slug path distal to valve V2. Valves V1 and V2 is then opened andthe sample solution is forced through the slug path such that it pushesthe reagent solution through the slug path into the reaction chamber.Typically the reaction chamber is filled by blind filling. As notedabove, the volume of reaction chamber exceeds the volume of reagentsolution forced into the chamber, with the result that both reagent andsample solutions are introduced into the chamber. It has been discoveredby the inventors that this process results in highly efficient mixing ofthe reagent and sample solutions. It has also been determined thatassays carried out using the FCS device system resulted in surprisinglysuperior results compared to use of prior art devices under the sameconditions (see, e.g., Example 7).

Efficiency in mixing for two solutions can be measured. For a firstsolution containing solute A and a second solution containing solute B,can be measured as the amount of B dispersed in the first solution at agiven period of time. For miscible solutions, the mixing will be 100%efficient over a long enough period of time. Efficiency can be measuredby art known methods. In one assay, mixing efficiency is assayed usingTaqMan Gene Expression Assays as an indicator. The assay includes a FAM™dye labeled TaqMan® MGB (minor groove binder) probe. The probe has beengenerally used as a quantification reporter in real time PCR.Fluorescence intensity in a microfluidic chamber corresponds to thepresence of the probe. In determining mixing efficiency, two solutionsare used. A first solution does not contain probe. A second solutioncontains 2 μM probe. The solutions are loaded into a microfluidic deviceand chamber loading initiated. Upon completion of loading the chamber(s)with the solutions, a fluorescent intensity image is taken by a highresolution fluorescence camera. That image is compared with a standardfluorescence image. The standard image is obtained by mixing the firstsolution with the second solution before loading the microfluidic systemand then loading the mixture into the microfluidic device. The mixingefficiency is defined as the fluorescence intensity of the on-devicemixed solutions divided by the intensity of the standard imageintensity. Using the devices and methods of the present invention,mixing occurs more rapidly than prior art devices. In one embodiment,twenty five percent (25%) efficiency is achieved in 30 minutes or less,often less than 20 minutes, often less than 10 minutes, often less than5 minutes, and sometimes less than 1 minute. In one embodiment, fiftypercent (50%) efficiency is achieved in 30 minutes or less, often lessthan 20 minutes, often less than 10 minutes, often less than 5 minutes,and sometimes less than 1 minute. In one embodiment, seventy fivepercent (75%) efficiency is achieved in 30 minutes or less, often lessthan 20 minutes, often less than 10 minutes, often less than 5 minutes,and sometimes less than 1 minute.

Without intending to be bound by a particular mechanism, it is believedthe superior results are a consequence of improved and highly efficientmixing of solutions achieved by the devices disclosed herein. Indeed,the mixing of the solutions is typically greater that 25% efficient,preferably greater than 35% efficient, more preferably greater than 50%efficient, more preferably greater that 65% efficient, more preferablygreater than 75% efficient, more preferably greater than 85% efficient,more preferably greater than 90% efficient, more preferably greater than95% efficient, more preferably greater that 99% efficient, and morepreferably about 100% efficient.

The volume of reagent displaced into the reaction chamber is determinedprimarily by the dimensions of the slug path and position of valves V1and V2. In general the volume of reagent introduced into the reactionchamber corresponds to the volume of the slug path lying between valvesV1 and V2, referred to as the “slug volume” (SV). The actual volume ofreagent introduced into the reaction chamber can be varied upward, ifdesired, based on design and process conditions. The careful reader willhave noted that the volume defined in each cell when valves V1, V2 andV3 of an array are closed exceeds the slug volume. This is illustratedin FIG. 4 in which the slug volume (SV) and the “non-flowing volume”(NFV), which is fluidically connected to the slug volume, are identifiedby cross-hatching. (All figures are for illustration and are not toscale.) If during the operation of the device the sample solution wasforced through the slug channel relatively slowly, a portion of thereagent solution in the NFV would diffuse into the reagent or samplesolution flowing past, increasing the amount of reagent introduced intothe reaction chamber. In practice, because flow through microfluidicchannels is primarily laminar the amount of solution that diffuses fromthe NFV into the flow path will usually be minor under conditions ofnormal use. Channel sizes, aspect ratios, and orientations, along withthe speed of flow of reagent and sample solutions through the slug path,can be adjusted to minimize, or if desired increase, the amount of NFVcontent that enters the reaction chamber.

In some FCS device, illustrated in FIGS. 7B and 7C, more than onereagent solution may be introduced along with sample into the reactionchamber. See Section VI(F) below.

The operation of an exemplary FCS device is illustrated in FIG. 3A-3H.The illustration in FIG. 3 is somewhat idealized in that it shows all ofthe reagent solution entering the reaction chamber before any of thesample solution enters. In practice, due to sheath flow, a bullet-shapedflow velocity profile will occur in the slug channel segment. Therefore,to achieve complete transfer of the reagent solution from the slug pathinto the reaction chamber, it is desirable that the reaction chambervolume be at least 2 times that of the slug volume (volume of solution 1introduced into the chamber). Preferably the reaction chamber volume isat least 3 times the slug volume, more preferably at least 4 times,often at least 5 times, at least 6 times, at least 7 times, at least 8times, or at least 9 times the slug volume.

FIG. 3A: Control channel 1 (260) is pressurized to close the valves thatfluidically isolate the ends of the slug channel segment (valves V1 andV2)

FIG. 3B: A reagent solution (solid dots) is introduced under pressurethrough the reagent input channels (300), through open valve 3 (V3), andthe slug channels are blind-filled.

FIG. 3C: Following the filling of the slug channels, control channel 2(270) is pressurized to actuate the valves (V3) that close off thereagent input channels (300) and thereby isolate the individual slugchannels from the other slug channels in the columns. In arrays in whichthere is a reagent bus line valve V3 can be located in the bus linebetween cells, or in the reagent input channel associated with eachcell.

FIG. 3D: Following the blind filling of the slug channels and theirisolation, a sample solution (open dots) is introduced under pressureinto each sample bus line (220). Although for clarity FIG. 3 showssequential addition of reagent and sample, it is also possible, andoften preferred, to inject reagent and sample at the same time, withvalves V1 and V2 closed and valve V3 open.

FIGS. 3E and 3F: The control channels 1 (260) are then depressurized toopen the interface valves (V1 and V2) that were previously closed toisolate the ends of the slug channels. The sample solution enters theslug channel at the first end and pushes the reagent into the reactionchamber. The conditions of the sample injection will vary. In someembodiments the sample solution is injected under pressure in the range8-15 PSI.

FIG. 3G: This results in a highly mixed, loaded reaction chamber (400)containing the 5 nL of reagent solution and 45 nL of sample solution (50nL total reaction chamber volume).

FIG. 3H: Finally, in this demonstration, control channel 1 ispressurized which results in the closure of the interface valves.

Although all rows in the reaction array and, accordingly, all sampleinput channels, are filled with the same same sample solution, there isno interconnection between the sample input channels of the individualrows and different samples can be introduced into the individual rows.In a 32×32 matrix having the configuration show in FIG. 1 (Exemplar 1)32 separate samples can be simultaneously mixed and loaded into reactionchambers with 32 separate reagents for 1024 individual experiments.

Although generally discussed in term of mixing of solutions, moregenerally the invention provides a method of combining two solutions ina microfluidic chamber. For example, the invention provides a method forcombining two solutions in a microfluidic reaction chamber byintroducing a predetermined volume of a first solution into a reactionchamber, introducing a predetermined volume of a second solution into areaction chamber, and fluidically isolating the reaction chamber.Advantageously, the FCS methods and devices result in introduction ofessentially all of the first solution (and a defined volume of thesecond solution) into a chamber. Following introduction into the chamberrapid mixing may occur due to an increased interface, as discussedabove, and because, for a solute in solution 1 the average diffusionalpath length to solution 2 is shorter than in prior art microfluidicdevices (and, equivalently, for a solute in solution 2 the averagediffusional path length to solution 1 is shorter than in prior artmicrofluidic devices). Thus, predetermined amounts of two solutions canbe introduced into a chamber having chamber volume specified herein inSection VII. The chamber can then be fluidically isolated.

Moreover, using methods described herein (see, e.g., FCS Device Exemplar5) more than two solutions can be introduced into a chamber bysequentially introducing predetermined volumes of N different solutionswhere N is at least 2. Usually N is from 2 to 10, usually 2-5, such as2, 3, 4 or 5. The combined total volume of the solutions is about equalto the fluid capacity of the reaction chamber.

V. SYSTEMS

The FCS device described herein may be used in conjunction withadditional elements including components external to the device.Examples of external components include external sensors, externalchromatography columns, actuators (e.g., pumps or syringes), controlsystems for actuating valves, data storage systems, reagent storageunits (reservoirs), detection and analysis devices (e.g., a massspectrophotometer), programmable readers, controllers, and othercomponents known in the art. See, e.g., co-pending and commonly ownedU.S. Patent Publication Nos. 2006/0006067, 2007/0074972; 2005/0214173;and 2005/0118073 each of which is incorporated herein for all purposes.

The microfluidic devices utilized in embodiments of the presentinvention may be further integrated into the carrier devices such as,for example, those described in co-pending and commonly owned U.S.Patent Application No. US2005/0214173A1, incorporated herein for allpurposes. These carriers provide on-board continuous fluid pressure tomaintain valve closure away from a source of fluid pressure, e.g., houseair pressure. Further provided is an automated system for charging andactuating the valves of the present invention as described therein. Ananother preferred embodiment, the automated system for chargingaccumulators and actuating valves employs a device having a platen thatmates against one or more surfaces of the microfluidic device, whereinthe platen has at least two or more ports in fluid communication with acontrolled vacuum or pressure source, and may include mechanicalportions for manipulating portions of the microfluidic device, forexample, but not limited to, check valves.

Another device utilized in embodiments of the present invention providesa carrier used as a substrate for stabilizing an elastomeric block.Preferably the carrier has one or more of the following features; a wellor reservoir in fluid communication with the elastomeric block throughat least one channel formed in or with the carrier; an accumulator influid communication with the elastomeric block through at least onechannel formed in or with the carrier; and, a fluid port in fluidcommunication with the elastomeric block, wherein the fluid port ispreferably accessible to an automated source of vacuum or pressure, suchas the automated system described above, wherein the automated sourcefurther comprises a platen having a port that mates with the fluid portto form an isolated fluid connection between the automated system forapplying fluid pressure or vacuum to the elastomeric block. In devicesutilized in certain embodiments, the automated source can also makefluid communication with one or more accumulators associated with thecarrier for charging and discharging pressure maintained in anaccumulator. In certain embodiments, the carrier may further comprise aregion located in an area of the carrier that contacts the microfluidicdevice, wherein the region is made from a material different fromanother portion of the carrier, the material of the region beingselected for improved thermal conduction and distribution propertiesthat are different from the other portion of the carrier. Preferredmaterials for improved thermal conduction and distribution include, butare not limited to silicon, preferably silicon that is highly polished,such as the type of silicon available in the semiconductor field as apolished wafer or a portion cut from the wafer, e.g., chip.

Embodiments of the present invention utilize a thermal source, forexample, but not limited to a PCR thermocycler, which may have beenmodified from its original manufactured state. Generally the thermalsource has a thermally regulated portion that can mate with a portion ofthe carrier, preferably the thermal conduction and distribution portionof the carrier, for providing thermal control to the elastomeric blockthrough the thermal conduction and distribution portion of the carrier.In a preferred embodiment, thermal contact is improved by applying asource of vacuum to a one or more channels formed within the thermallyregulated portion of the thermal source, wherein the channels are formedto contact a surface of the thermal conduction and distribution portionof the carrier to apply suction to and maintain the position of thethermal conduction and distribution portion of the carrier. In apreferred embodiment, the thermal conduction and distribution portion ofthe carrier is not in physical contact with the remainder of thecarrier, but is associated with the remainder of the carrier and theelastomeric block by affixing the thermal conduction and distributionportion to the elastomeric block only and leaving a gap surrounding theedges of the thermal conduction and distribution portion to reduceparasitic thermal effects caused by the carrier. It should be understoodthat in many aspects of the invention described herein, the preferredelastomeric block could be replaced with any of the known microfluidicdevices in the art not described herein, for example devices producedsuch as the GeneChip® by Affymetrix® of Santa Clara, Calif., USA, or byCaliper of Mountain View, Calif., USA. U.S. patents issued to Soane,Parce, Fodor, Wilding, Ekstrom, Quake, or Unger, describe microfluidicor mesoscale fluidic devices that can be configured to utilize the carryslug mixing methods or devices of the current invention. A unit cell ofthe invention can be used as a mixing module in a microfluidic devicecontaining other elements. In such an embodiment the reagent inputchannel 300 and/or sample input channel 290 may be linked to a solutionreservoir or, alternatively, to a channel that is an output of adifferent on-chip element such as a column, chamber, or channel.Similarly, the reaction chamber may include an exit channel (500) thatfluidically connected to a different on-chip element such as a column,chamber, or channel. Examples include microfluidic proteincrystallization devices, bioprocessing devices including cell-basedassay devices, microfluidic immunoassay devices, combinatorial synthesissystems, nucleic acid sample preparation devices, electrophoreticanalytical devices, microfluidic microarray devices, microfluidicdevices incorporating electronic or optical sensors, and nucleic acidand protein sequencing devices.

VI. FCS DEVICE EXEMPLARS

A. FCS Device Exemplar 1

FIG. 1 is a diagram illustrating a portion of the array of an FCSdevice. This design has been used to fabricate a 32-column by 32-row FCSarray having a footprint less than 10 cm² and a height of 4 mm.

In operation of the Exemplar 1 (“E1”) FCS device, control channel 1(260) is pressurized to close valves V1 and V2. Reagent solution isflowed through reagent input channels (300) and thereby introduced intothe slug path (250) of the cell. Because reagent input channels (300)are fluidically linked to the slug channel of an adjacent cell in acolumn, infusion of reagent solution through the reagent input channelof one cell fills all cells in the column. When the fill step iscompleted, valve V3 can be closed. Concurrent with, prior to, orsubsequent to the loading of the reagent solution, sample solution isloaded via sample bus line (220), through fluid communication via, andinto the distal portion of the slug path, and maintained under pressureagainst valve V2. Valves V2 and V3 are then opened and sample solution,fed by the sample bus line (220) flows through the via into the slugpath to reaction chamber 400. As the sample solution moves through theslug path it pushes (displaces) the reagent solution out of the slugpath and into the reaction chamber 400 where it is mixed with the samplesolution initiating the reaction of interest.

In the FCS device illustrated in FIGS. 1 and 2, the first elastomericlayer contains elements 220, 260, and 270 and the second elastomericlayer contains elements 250, 300, and 400.

B. FCS Device Exemplar 2

FIG. 4 is a diagram illustrating a portion of the array of an FCSdevice. This design has been used to fabricate a 48-column by 48-row FCSarray having a footprint less than 10 cm² and a height of 4 mm.

In operation of the Exemplar 2 (“E2”) FCS device, control channel 1(260) is pressurized to close valves V1 and V2. Reagent solution isflowed through the reagent bus line (230) and reagent input channels(300) and thereby introduced into the slug path (250) of the cell. Whenthe fill step is completed, valve V3 can be closed. Concurrent with,prior to, or subsequent to the loading of the reagent solution, samplesolution is loaded via sample bus line (220), through fluidcommunication via 240, and into the distal portion of the slug path, andmaintained under pressure against valve V2. Valves V2 and V3 are thenopened and sample solution, fed by the sample bus line (220) flowsthrough fluid communication via 240 through the slug path to reactionchamber 400. As the sample solution moves through the slug path itpushes (displaces) the reagent solution out of the slug path and intothe reaction chamber 400 where it is mixed with the sample solutioninitiating the reaction of interest.

In the FCS device illustrated in FIG. 4, the first elastomeric layercontains elements 220, 260, and 270 and the second elastomeric layercontains elements 230, 250, 300, and 400.

C. FCS Device Exemplar 3

FIG. 5 is a diagram illustrating a cell of an array of an FCS device. Inthe operation of the Exemplar 3 (“E3”) FCS device, control channel 1(260) is pressurized to close push-down valves V1 and V2. Reagentsolution is flowed through a reagent bus line (230), through a valvedreagent input line (300), and introduced into the slug path (250) of thecell. When the fill step is completed, valve V3 can be closed.Concurrent with, prior to, or subsequent to the loading of the reagentsolution, sample solution is loaded via sample bus line (220), throughfluid communication via 240, and into the distal portion of the slugpath, and maintained under pressure against valve V2. Valves V2 and V3are then opened and sample solution, fed by the sample bus line (220)flows through the slug path pushing (displacing) the reagent solutionthrough fluid communication via 280 into reaction chamber 400, with aportion of the sample solution also entering the reagent chamber,whereupon the reagent solution and the sample solution are mixed.

In the FCS device illustrated in FIG. 5, an upper elastomeric layercontains elements 220, 260, and 270, a middle elastomeric layer containselements 230, 250 and 300, and a lower elastomeric layer containschamber 400.

D. FCS Device Exemplar 4

FIG. 6 is a diagram illustrating selected elements of one cell of anarray of an FCS device (Exemplar 4, “E4”). In the E4 FCS device the slugpath has channel segments in two different layers of a multilayerelastomeric device. As illustrated in the figure, the slug pathcomprises a first channel segment (250 a) in one layer of the device, asecond channel segment (250 c) in a third layer of the device, and afluid communication via (250 b) linking the channel segments. Forclarity the fluid communication via is represented in the figure by anarrow. This design has been used to fabricate a 48-column by 96-row FCSarray having a footprint less than 10 cm² and a height of 4 mm.

In operation of the Exemplar 4 FCS device, control channel 1 (260) ispressurized to close valves V1 and V2. In the embodiment shown in thisfigure, the valves actuated by control channel 260 are“push-up/push-down valve” is used to simultaneously closes valve V1 inchannel segment 260 c and valve V2 in 260 a. A push-up/push-down valvecan be created, for example and not limitation, in an elastomeric deviceby sandwiching a control line between two fluid channels as illustratedin FIG. 8. Layer C comprises a control line (in this case, control line260), while layers B and D are fluidic channels (in this case channelsegment 250 a and 250 c). As can be seen in the figure, the bottomsurface of the fluid channel in layer D is defined by a thin uppermembrane in layer C. This thin upper membrane comprises a push-up valvewhen the control line is pressurized, since the membrane would deflectupwards sealing off the fluid channel in layer D. Similarly, the uppermembrane of the fluid channel in layer B defines the lower surface ofthe control channel in layer C. When the control channel is pressurized,this thin membrane deflects downwards creating a push-down valve. Thepush-up/push-down configuration allows valves to be stacked on top ofeach other. This allows for designs with very high feature density.

With valves V1 and V2 closed, reagent solution is flowed through reagentbus line 230 into reagent input channel 300 and introduced into the slugpath (250 abc). Slug path 250 comprises two slug channel segments (250a, lower, and 250 c, upper) which are linked by a fluid communicationvia (250 b). In some embodiments the region of slug channel segment 250c that lies directly over fluid communication via (250 b) is enlarged toincrease the slug volume. For clarity, this enlargement is notillustrated in FIG. 6. As a consequence of these steps reagent solutionfills the slug path 250 abc. Concurrent with this step, sample solutionis loaded via sample bus line (220) into the sample input channel (290)and maintained under pressure against valve V2. Valve V3 is then closedby pressurizing control channel 270.

Control channel 1 (260) is then depressurized to open valves V1 and V2.Sample solution, fed by the sample bus line (220) then flows through thevalve into slug channel segment 250 a, through fluid communication via250 b, into slug channel segment 250 c, and into reaction chamber 400.As the sample solution moves through the slug path it pushes (displaces)the reagent solution out of the slug path and into the reaction chamber400 where it is mixed with the sample solution. Following the push,valve V2 is closed. Most often, valves V1 and V2 are both closed. Insome embodiments a reaction is initiated by the mixing the reagent andsample solutions. In some embodiments the reaction is initiated byanother stimulus, most typically a change in temperature of the reactionchamber. An example is the application of heat to initiate a nucleicacid amplification reaction.

In the FCS device illustrated in FIG. 6, the first elastomeric layercontains elements 220, 290, and 250 c; the second elastomeric layercontains elements 260, 270 and 250 b (which traverses the second layer);and the third layer contains elements 230, 300, 250 c and 400. As shown,reaction chamber 400 spans several layers to provide the desired volume.

FCS devices having slug channel segments on an even greater number oflayers are contemplated. For example, a high density array may have slugchannel segments on four or more levels (e.g., four horizontal channelslinked by three fluid communication vias). In such a device fluid canflow through the slug flow path in multiple horizontal planes andmultiple vertical planes, and in both vertical directions (up and down)during operation of the device.

F. FCS Device Exemplar 5

FIG. 7B-C illustrate a FCS unit design that provides for mixing ofmultiple metered solutions. FIG. 7A shows selected elements of unitcells previously discussed (control channels and reagent bus line arenot shown). FIG. 7B shows a design for mixing two reagent solutions(“reagent 1” and “reagent 2”) and a sample solution. As discussed suprain Section II, the designations “sample” and “reagent” are forconvenience and do not necessarily describe the nature of the solutions.In FIG. 7B, slug channel 250 has two segments, 250 a and 250 b definedand bounded by valves V1 and V2, and V2 and V4, respectively. Withvalves V3 and V5 open, reagent 1 is instilled into segment 250 a andreagent 2 is instilled into segment 250 b. The flow channel segments canbe filled simultaneously or in any order. Sample solution is flowedthrough sample bus line 200 into segment 290 and maintained underpressure against valve V4. Valves V3 and V5 are closed and valves V1, V2and V4 are opened. Sample solution under pressure flows through slugpath 250 pushing reagent solutions 1 and 2 into the reaction chamber400. FIG. 7C illustrates an analogous design in which up to 5 solutionsmay be combined. See Example 4, infra. The fluid capacity of reactionchamber 400 should exceed the combined fluid volumes of the reagentsolutions pushed into the chamber.

G. FCS Devices with Check Valves

In some embodiments the unit cells of a FCS device comprises one or moremicrofluidic check valves. A microfluidic check valve is a valve thatallows solution to flow in only one direction through the valve. Avariety of check valves are known. See, e.g., Adams et al., 2005, J.Micromech. Microeng. 15:1517-21 and references 6-12 therein. In oneembodiment the check valve has a small dead volume (e.g., less than 100nL) and comprises an outlet chamber in fluidic communication with anoutlet channel, an inlet chamber in fluidic communication with an inletchannel, and a deflectable membrane between the outlet chamber and theinlet chamber, the membrane having a fluidic channel that places theinlet chamber in fluidic communication with the outlet chamber. Anexemplary check valve is described in copending applicationPCT/US07/80489 (filed Oct. 4, 2007) the entire content of which isincorporated by reference. An exemplary check valve is described belowin Example 6 and illustrated in FIGS. 10 and 11.

In certain embodiments the check valve is located in the analysis unitin the slug path between valve V1 and the reaction chamber 400, as isillustrated in FIG. 9A. Inclusion of the check valve proximal to thereaction chamber provides certain advantages. For example, in operationof an FCS device, after reagent and sample solutions are delivered tothe reaction chamber, the chamber is often isolated, e.g., by closingvalve V1, so that the reaction is contained in the reaction chamber. Byusing a check valve the reaction chamber contents may be effectivelycontained in the chamber without the necessity of closing valve V1and/or without the need to maintain valve V1 in the closed state for theduration of the reaction and/or duration of any analysis steps. This isespecially useful when the FCS device is physically moved after thereaction chamber is filled (e.g., moved to a thermocycler or reader). Inan alternative embodiment, a check valve can be placed distal to valve 1as shown in FIG. 9B.

H. FCS Devices Having Reaction Chambers with Exit Channels

As illustrated in FIG. 9C in some embodiments the reaction chamber be indirect fluidic communication with one or more than one flow channels(e.g., flow channel 500) in addition to the slug path. Such additionalflow channels are valved (e.g., exit valve VEX).

VII. CHARACTERISTICS AND FABRICATION OF FCS DEVICES

FCS devices of the invention can be constructed out of any material orcombination of materials that can be fabricated to have microfluidicchannels and chambers, and valves that regulate flow through channelsand into chambers. Materials from which a device can be fabricatedinclude, without limitation, elastomers, silicon, glass, metal, polymer,ceramic, inorganic materials, and/or combinations of these materials.

The methods used in fabrication of a FCS device will vary with thematerials used, and include soft lithography methods, microassembly,bulk micromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,modular assembly methods, replica molding methods, injection moldingmethods, hot molding methods, laser ablation methods, combinations ofmethods, and other methods known in the art or developed in the future.A variety of exemplary fabrication methods are described in Fiorini andChiu, 2005, “Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; U.S.Pat. No. 6,767,706 B2, e.g., Section 6.8 “Microfabrication of a SiliconDevice”; Terry et al., 1979, A Gas Chromatography Air AnalyzerFabricated on a Silicon Wafer, IEEE Trans. on Electron Devices, v.ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis Systems,New York, Kluwer; Webster et al., 1996, Monolithic Capillary GelElectrophoresis Stage with On-Chip Detector in International ConferenceOn Micro Electromechanical Systems, MEMS 96, pp. 491-496; andMastrangelo et al., 1989, Vacuum-Sealed Silicon MicromachinedIncandescent Light Source, in Intl. Electron Devices Meeting, IDEM 89,pp. 503-506.

A) Elastomeric Fabrication

In preferred embodiments, the device is fabricated using elastomericmaterials. Fabrication methods using elastomeric materials and methodsfor design of devices and their components have been described in detailin the scientific can patent literature. See, e.g., Unger et al., 2000,Science 288:113-16; U.S. Pat. Nos. 6,960,437 (Nucleic acid amplificationutilizing microfluidic devices); 6,899,137 (Microfabricated elastomericvalve and pump systems); 6,767,706 (Integrated active flux microfluidicdevices and methods); 6,752,922 (Microfluidic chromatography); 6,408,878(Microfabricated elastomeric valve and pump systems); 6,645,432(Microfluidic systems including three-dimensionally arrayed channelnetworks); U.S. Patent Application publication Nos. 2004/0115838,20050072946; 20050000900; 20020127736; 20020109114; 20040115838;20030138829; 20020164816; 20020127736; and 20020109114; PCT patentpublications WO 2005/084191; WO05030822A2; and WO 01/01025; Quake &Scherer, 2000, “From micro to nanofabrication with soft materials”Science 290: 1536-40; Xia et al., 1998, “Soft lithography” AngewandteChemie-International Edition 37:551-575; Unger et al., 2000, “Monolithicmicrofabricated valves and pumps by multilayer soft lithography” Science288:113-116; Thorsen et al., 2002, “Microfluidic large-scaleintegration” Science 298:580-584; Chou et al., 2000, “MicrofabricatedRotary Pump” Biomedical Microdevices 3:323-330; Liu et al., 2003,“Solving the “world-to-chip” interface problem with a microfluidicmatrix” Analytical Chemistry 75, 4718-23,” Hong et al, 2004, “Ananoliter-scale nucleic acid processor with parallel architecture”Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005, “Disposablemicrofluidic devices: fabrication, function, and application”Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidic tectonics: acomprehensive construction platform for microfluidic systems.” Proc.Natl. Acad. Sci. USA 97:13488-13493; Rolland et al., 2004,“Solvent-resistant photocurable “liquid Teflon” for microfluidic devicefabrication” J. Amer. Chem. Soc. 126:2322-2323; Rossier et al., 2002,“Plasma etched polymer microelectrochemical systems” Lab Chip 2:145-150;Becker et al., 2002, “Polymer microfluidic devices” Talanta 56:267-287;Becker et al., 2000, and other references cited herein and found in thescientific and patent literature.

i. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels preferably have width-to-depthratios of about 10:1. A non-exclusive list of other ranges ofwidth-to-depth ratios in accordance with embodiments of the presentinvention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels have widths of about 1 to 1000 microns. Anon-exclusive list of other ranges of widths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels have depths of about 1 to 100 microns. A non-exclusivelist of other ranges of depths of flow channels in accordance withembodiments of the present invention is 0.01 to 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250 microns, andmore preferably 1 to 100 microns, more preferably 2 to 20 microns, andmost preferably 5 to 10 microns. Exemplary channel depths includeincluding 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, 2 μm,3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm, 22.5μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, and 250μm.

Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, a layer is 50 microns to over a centimeter thick,and more preferably approximately 4 mm thick. A non-exclusive list ofranges of thickness of the elastomer layer in accordance with otherembodiments of the present invention is between about 0.1 micron to 1cm, 1 micron to 1 cm, 10 microns to 0.5 cm, 100 microns to 10 mm.

Accordingly, membranes separating flow channels have a typical thicknessof between about 0.01 and 1000 microns, more preferably 0.05 to 500microns, more preferably 0.2 to 250, more preferably 1 to 100 microns,more preferably 2 to 50 microns, and more preferably 5 to 40 microns,and most preferably 10-25 μm. Exemplary membrane thicknesses include0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20 μm,22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

ii. Reaction Chambers

Reaction chamber dimensions in an FCS device can vary over a broadrange. In embodiments of the present invention, reaction volumes rangingfrom 10 picoliters to 100 nanoliters are utilized. In some embodiments,reaction volumes greater than 100 nanoliters are utilized. Reactionchambers may also be in the microliter, nanoliter, picoliter, femtoliteror lower range of volume. In one embodiment, the reaction chamber volumeis between 1-1000 femtoliters. Merely by way of example, in anembodiment, the methods and systems of the present invention areutilized with reaction volumes of 10 picoliters, 50 picoliters, 100picoliters, 250 picoliters, 500 picoliters, and 1 nanoliter. Inalternative embodiments, reaction volumes of 2 nanoliters, 5 nanoliters,10 nanoliters, 20 nanoliters, 30 nanoliters, 40 nanoliters, 50nanoliters, 75 nanoliters, and 100 nanoliters are utilized. In anotherembodiment, the reaction chamber volume is between 1-1000 picoliters. Inanother embodiment, the reaction chamber volume is between 0.01-100nanoliters, preferably between 1-75 nanoliters. In one embodiment thereaction chamber volume is about 50 nanoliters. In one embodiment thereaction chamber volume is about 7.6 nanoliters. In another embodiment,the reaction chamber volume is 6 nL. The volume defined for the firstsolution in the flow channel (the slug volume or carry-on volume) is afraction of the reaction chamber volume. In various embodiments, thefraction may be ⅞, ¾, ⅝, ½, ⅜, ¼, ⅕, ⅛, 1/10, 1/12, 1/20, 1/25, 1/50,1/100, or less of the total reaction chamber volume. Preferably thefraction is less than ½, more preferably less than ¼, more preferablyless than ⅛. In some embodiments the volume of reagent solution is about1/10th the volume of the reaction chamber and the volume of the samplesolution is about 9/10th of the volume of the reaction chamber.

Reaction chambers are often cuboid due in part to relative ease ofmanufacture, however other shapes can be used. In preferred embodimentsthe chamber comprises internal edges (i.e., is not spherical). Theseedges enhance mixing of reagent and sample. A cuboid chamber has 12internal edges. In one embodiment the reagent chamber has at least 2internal edges (e.g., a cylinder). More often the chamber has at least10, at least 12, at least 14, at least 16, or at least 20 internaledges.

iii. Elastomeric Valves

As discussed above, in preferred embodiments the FCS device compriseselastomeric materials and monolithic valves, such as a pressure-actuated“elastomeric valve.” A pressure-actuated elastomeric valve consists of aconfiguration in which two microchannels are separated by an elastomericsegment that can be deflected into or retracted from one of the channels(e.g., a flow channel) in response to an actuation force applied to theother channel (e.g., a control channel). Examples of elastomeric valvesinclude upwardly-deflecting valves (see, e.g., US 20050072946),downwardly deflecting valves (see, e.g., U.S. Pat. No. 6,408,878), sideactuated valves (see, e.g., US 20020127736, e.g., paragraphs 0215-0219),normally-closed valves (see, e.g., U.S. Pat. Nos. 6,408,878 B2 and6,899,137) and others. In some embodiments a device can have acombination of valves (e.g., upwardly deflecting valves and downwardlydeflecting valves). Valves can be actuated by injecting gases (e.g.,air, nitrogen, and argon), liquids (e.g., water, silicon oils,perfluoropolyalkylether, and other oils), solutions containing saltsand/or polymers (including but not limited to polyethylene glycol,glycerol and carbohydrates) and the like into the control channel. Somevalves can be actuated by applying a vacuum to the control channel.

iv. Multilayer Soft Lithography Construction Techniques and Materials

The microfluidic devices disclosed herein are typically constructed atleast in part from elastomeric materials and constructed by single andmultilayer soft lithography (MSL) techniques and/or sacrificial-layerencapsulation methods (see, e.g., Unger et al., 2000, Science288:113-116, and PCT Publication WO 01/01025, both of which areincorporated by reference herein in their entirety for all purposes).Utilizing such methods, microfluidic devices can be designed in whichsolution flow through flow channels of the device is controlled, atleast in part, with one or more control channels that are separated fromthe flow channel by an elastomeric membrane or segment. This membrane orsegment can be deflected into or retracted from the flow channel withwhich a control channel is associated by applying an actuation force tothe control channels. By controlling the degree to which the membrane isdeflected into or retracted out from the flow channel, solution flow canbe slowed or entirely blocked through the flow channel. Usingcombinations of control and flow channels of this type, one can preparea variety of different types of valves and pumps for regulating solutionflow as described in extensive detail in Unger et al., 2000, Science288:113-116, PCT Publications WO/02/43615 and WO 01/01025, and otherreferences cited herein and known in the art.

Soft Lithographic Bonding:

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In one aspect, the various layers of elastomer are bound together in aheterogenous bonding in which the layers have a different chemistry.Alternatively, a homogenous bonding may be used in which all layerswould be of the same chemistry. Thirdly, the respective elastomer layersmay optionally be glued together by an adhesive instead. In a fourthaspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Elastomeric layers may be created by spin-coating an RTV mixture onmicrofabricated mold at 2000 rpm for 30 seconds yielding a thickness ofapproximately 40 microns. Additional elastomeric layers may be createdby spin-coating an RTV mixture on microfabricated mold. Both layers maybe separately baked or cured at about 80° C. for 1.5 hours. Theadditional elastomeric layer may be bonded onto first elastomeric layerat about 80° C. for about 1.5 hours.

Suitable Elastomeric Materials:

Allcock et al, Contemporary Polymer Chemistry, 2nd Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, elastomeric layers maypreferably be fabricated from silicone rubber. However, other suitableelastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones. Anon-exclusive list of elastomeric materials which may be utilized inconnection with the present invention includes polyisoprene,polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and siliconepolymers; or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),perfluoropolyalkylether siloxane block copolymer,poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)(nitrile rubber), poly(1-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoro-ethylene (Teflon).

a. Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

b. Polyisobutylene:

Pure polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thepolyisobutylene backbone, which may then be vulcanized as above.

c. Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

d. Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

e. Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

Cross Linking Agents:

In addition to the use of the simple “pure” polymers discussed above,crosslinking agents may be added. Some agents (like the monomers bearingpendant double bonds for vulcanization) are suitable for allowinghomogeneous (A to A) multilayer soft lithography or photoresistencapsulation; in such an approach the same agent is incorporated intoboth elastomer layers. Complementary agents (i.e. one monomer bearing apendant double bond, and another bearing a pendant Si—H group) aresuitable for heterogeneous (A to B) multilayer soft lithography. In thisapproach complementary agents are added to adjacent layers.

Other Materials:

In addition, polymers incorporating materials such as chlorosilanes ormethyl-, ethyl-, and phenylsilanes, and polydimethylsiloxane (PDMS) suchas Dow Chemical Corp. Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical may also be used.

Doping and Dilution:

Elastomers may also be “doped” with uncrosslinkable polymer chains ofthe same class. For instance RTV 615 may be diluted with GE SF96-50Silicone Fluid. This serves to reduce the viscosity of the uncuredelastomer and reduces the Young's modulus of the cured elastomer.Essentially, the crosslink-capable polymer chains are spread furtherapart by the addition of “inert” polymer chains, so this is called“dilution”. RTV 615 cures at up to 90% dilution, with a dramaticreduction in Young's modulus.

Other examples of doping of elastomer material may include theintroduction of electrically conducting or magnetic species, asdescribed in detail below in conjunction with alternative methods ofactuating the membrane of the device. Should it be desired, doping withfine particles of material having an index of refraction different thanthe elastomeric material (i.e. silica, diamond, sapphire) is alsocontemplated as a system for altering the refractive index of thematerial. Strongly absorbing or opaque particles may be added to renderthe elastomer colored or opaque to incident radiation, which may be ofbenefit in an optically addressable system.

Finally, by doping the elastomer with specific chemical species, thesedoped chemical species may be presented at the elastomer surface, thusserving as anchors or starting points for further chemicalderivitization.

vi. Vent Channels

In some embodiments, the FCS device has channels, referred to as “ventchannels” positioned to accelerate or facilitate withdrawal of gas fromthe reaction chamber or channels to facilitate filling (e.g., dead-endor blind filling). See PCT Publication WO 2006/071470, incorporatedherein by reference. A vent channel system comprises channels separatedfrom, e.g., a sample (or reagent) bus line by a thin gas permeable(e.g., elastomeric) membrane. The vent channels typically lie over orunder a bus line (e.g., in a vent layer or control layer). Vapor andgasses are expelled out of the bus line by passing through anintervening gas permeable material (such as an elastomer), and entersthe vent channels(s). Vapor and gasses can diffuse into the vent channelor removal can be accelerated by reducing the pressure in the ventchannel relative to the bus line. This reduction can be achieved, forexample, by flowing dry gas (e.g., air or N₂) through the ventchannel(s) or drawing a vacuum through the channel(s), or by any othermethod that reduces vent channel pressure (including reduction caused byBernoulli's principle).

The dimensions of vent channels can vary widely. In an exemplary aspect,vent channels have at least one cross-sectional dimension in the rangeof 0.05 to 1000 microns, often 10 to 500 microns, and most often 50 to200 microns. In some embodiments, the channel height is not more thanabout 500 microns or less than about 20 microns (in some embodiments,not more than about 250 microns or less than about 50 microns) and thechannel width is not more than 5000 microns or less than 20 microns). Inone embodiment, vent channels have rectangular cross-sectionaldimensions of about 15 microns×50 microns. In some embodiments, ventchannels preferably have width-to-depth ratios of about 1:10 to 100:1,such as between about 2:1 and 1:2, and sometimes about 1:1. Inembodiments in which a vacuum is applied to a vent channel dimensionsmay be selected to avoid collapse of the channel under vacuum (e.g.,higher height:width ratios). However, the vent channels are not limitedto these particular dimensions or proportions.

As noted above, in some embodiments, the lumen of the vent channel(s) isseparated from the interior of the bus line by less than 1000 microns,such as from 0.05 to 1000 microns, often from 1 to 500 microns, oftenfrom 1 to 200 microns, and most often from 5 to 50 microns. In oneembodiment, a vent is placed below the sample bus line consisting of agroup of six 15×50 micron channels separated from the bus line by a 15micron membrane (gas-permeable). In another embodiment the bus linehexfurcates into six parallel lines (each 50 microns wide) that crossover the six vent lines, thus increasing the amount of membrane area tofacilitate vapor and/or gas expulsion

With reference to an elastomeric or partially elastomeric device, asystem of vent channel can lie in an elastomer layer one side of whichconstitutes a portion of the interior surface of the bus line. Forexample, in a “wholly” elastomeric device the vent channels may lie inthe elastomer layer above or below the flow channel layer (and, fordevices with control channels, on the side of the flow layer oppositethe control channel layer or in the control channel layer). Ventchannels may also be incorporated into the flow channel layer. In someembodiments, providing vent channels above the bus line is the optimalarrangement. However, it is generally easier to fabricate an MSL chipwith the vent below the bus line (e.g., as part of the control layer).

vii. Characteristics and Fabrication of Hybrid and Non-Elastomeric FCSDevices

As noted, a variety of materials can be used in fabrication of the FCSdevice. Devices can be fabricated from combinations of materials. In ahybrid device channels and/or the reaction chamber may be formed from anon-elastomeric substrate, but the channels and/or the reaction chamberhave an elastomeric component sufficient that allows the chambers orreaction channels to be blind filled. For example, in some embodimentsthe walls and ceiling of a reaction chamber and/or flow channels areelastomeric and the floor of the reactor is formed from an underlyingnonelastomeric substrate (e.g., glass), while in other embodiments, boththe walls and floors of the reaction chamber and/or flow channels areconstructed from a nonelastomeric material, and only the ceiling of thereaction chamber and/or flow channels is constructed from elastomer.These channels and chambers are sometimes referred to as “compositestructures.” See, e.g., US 20020127736. A variety of approaches can beemployed to seal the elastomeric and nonelastomeric components of adevice, some of which are described in U.S. Pat. No. 6,719,868 and US20020127736, paragraph [0227] et seq.

Valves of various types are known in the art, including micromechanicalvalves, elastomeric valves, solid-state microvalves, and others. See,e.g., Felton, 2003, The New Generation of Microvalves” AnalyticalChemistry 429-432. Two common approaches to fabrication ofmicroelectromechanical (MEMS) structures such as pumps and valves aresilicon-based bulk micro-machining (which is a subtractive fabricationmethod whereby single crystal silicon is lithographically patterned andthen etched to form three-dimensional structures), and surfacemicro-machining (which is an additive method where layers ofsemiconductor-type materials such as polysilicon, silicon nitride,silicon dioxide, and various metals are sequentially added and patternedto make three-dimensional structures).

In addition to elastomeric valves actuated by pressure-based actuationsystems, monolithic valves with an elastomeric component andelectrostatic, magnetic, electrolytic and electrokinetic actuationsystems may be used. See, e.g., US 20020109114; US 20020127736, e.g., at¶¶ 0168-0176; and U.S. Pat. No. 6,767,706 B2 e.g., at §6.3. Likewiseother types of valves are known in the art and may be used. See, e.g.Jeon et al. U.S. Pat. No. 6,767,194, incorporated herein by reference,and Luo et al. 2003, “Monolithic valves for microfluidic chips based onthermoresponsive polymer gels” Electrophoresis 24:3694-3702. Each of theaforementioned references is incorporated herein by reference.

VIII. EXEMPLARY REACTIONS

The devices and methods of the invention are useful for any microfluidicprocess that involves combining mixing two or more solutions. A numberof reactions useful for detection, quantitation and analysis of nucleicacids are described below in this section. However, the uses of the FCSdevice are not limited to “reactions” of this type. Other “reactions”include, but are not limited to, binding interactions (e.g.,ligand-antiligand interactions, including antibody-antigen interactions,avidin-biotin interactions), protein-ligand interactions andinteractions between cells and various compounds, trapping, chemical orbiochemical synthesis, analysis of cells or viruses, and others.

Nucleic acid amplification reactions can be carried out using FCSdevices and methods. For example, devices of the invention may bedesigned to conduct thermal cycling reactions. PCR is perhaps the bestknown amplification technique. The devices utilized in embodiments ofthe present invention are not limited to conducting PCR amplifications.Other types of amplification reactions that can be conducted include,but are not limited to, (i) ligase chain reaction (LCR) (see Wu andWallace, Genomics 4:560 (1989) and Landegren et al., Science 241:1077(1988)); (ii) transcription amplification (see Kwoh et al., Proc. Natl.Acad. Sci. USA 86:1173 (1989)); (iii) self-sustained sequencereplication (see Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874(1990)); and (iv) nucleic acid based sequence amplification (NASBA)(see, Sooknanan, R. and Malek, L., BioTechnology 13: 563-65 (1995)).Each of the foregoing references is incorporated herein by reference intheir entirety for all purposes.

Amplification products (amplicons) can be detected and distinguished(whether isolated in a reaction chamber or at any subsequent time) usingroutine methods for detecting nucleic acids. Many different signalmoieties may be used in various embodiments of the present invention.For example, signal moieties include, but are not limited to,fluorophores, radioisotopes, chromogens, enzymes, antigens, heavymetals, dyes, phosphorescence groups, chemiluminescent groups, minorgrove binding probes, and electrochemical detection moieties. Exemplaryfluorophores that may be used as signal moieties include, but are notlimited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein,VIC™, LIZ™, Tamra™, 5-FAM™, 6-FAM™, and Texas Red (Molecular Probes).(VIC™, LIZ™, Tamra™, 5-FAM™, and 6-FAM™ (all available from AppliedBiosystems, Foster City, Calif.). Exemplary radioisotopes include, butare not limited to, ³²P, ³³P, and ³⁵S. Signal moieties also includeelements of multi-element indirect reporter systems, e.g.,biotin/avidin, antibody/antigen, ligand/receptor, enzyme/substrate, andthe like, in which the element interacts with other elements of thesystem in order to effect a detectable signal. Certain exemplarymulti-element systems include a biotin reporter group attached to aprobe and an avidin conjugated with a fluorescent label. Detailedprotocols for methods of attaching signal moieties to oligonucleotidescan be found in, among other places, G. T. Hermanson, BioconjugateTechniques, Academic Press, San Diego, Calif. (1996) and S. L. Beaucageet al., Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons,New York, N.Y. (2000).

Amplicons comprising double-stranded DNA can be detected usingintercalation dyes such as SYBR™, Pico Green (Molecular Probes, Inc.,Eugene, Oreg.), ethidium bromide and the like (see Zhu et al., 1994,Anal. Chem. 66:1941-48) and/or gel electrophoresis. More often,sequence-specific detection methods are used (i.e., amplicons aredetected based on their nucleotide sequence). Examples of detectionmethods include hybridization to arrays of immobilized oligo orpolynucleotides, and use of differentially labeled molecular beacons orother “fluorescence resonance energy transfer” (FRET)-based detectionsystems. FRET-based detection is a preferred method for detectionaccording to some embodiments of the present invention. In FRET-basedassays a change in fluorescence from a donor (reporter) and/or acceptor(quencher) fluorophore in a donor/acceptor fluorophore pair is detected.The donor and acceptor fluorophore pair are selected such that theemission spectrum of the donor overlaps the excitation spectrum of theacceptor. Thus, when the pair of fluorophores are brought withinsufficiently close proximity to one another, energy transfer from thedonor to the acceptor can occur and can be detected. A variety of assaysare known including, for example and not limitation, template extensionreactions, quantitative RT-PCR, Molecular Beacons, and Invader assays,these are described briefly below.

FRET and template extension reactions utilize a primer labeled with onemember of a donor/acceptor pair and a nucleotide labeled with the othermember of the donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during an template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods areparticularly useful in conducting single base pair extension reactionsin the detection of single nucleotide polymorphisms and are described inU.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719. The reactionscan optionally be thermocycled to increase signal using the temperaturecontrol methods and apparatus described throughout the presentspecification.

A variety of so-called “real time amplification” methods or “real timequantitative PCR” methods can also be used to determine the quantity ofa target nucleic acid present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantitation method which can be used successfully with the devicesdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan”method. See, for example, U.S. Pat. No. 5,723,591.

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye. In solution, the two end sectionscan hybridize with each other to form a hairpin loop. In thisconformation, the reporter and quencher dye are in sufficiently closeproximity that fluorescence from the reporter dye is effectivelyquenched by the quencher dye. Hybridized probe, in contrast, results ina linearized conformation in which the extent of quenching is decreased.Thus, by monitoring emission changes for the two dyes, it is possible toindirectly monitor the formation of amplification product. Probes ofthis type and methods of their use are described further, for example,by Piatek et al., 1998, Nat. Biotechnol. 16:359-63; Tyagi, and Kramer,1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat.Biotechnol. 16:49-53 (1998).

The Scorpion detection method is described, for example, by Thelwell etal. 2000, Nucleic Acids Research, 28:3752-3761 and Solinas et al., 2001,“Duplex Scorpion primers in SNP analysis and FRET applications” NucleicAcids Research 29:20. Scorpion primers are fluorogenic PCR primers witha probe element attached at the 5′-end via a PCR stopper. They are usedin real-time amplicon-specific detection of PCR products in homogeneoussolution. Two different formats are possible, the stem-loop format andthe duplex format. In both cases the probing mechanism isintramolecular. The basic elements of Scorpions in all formats are: (i)a PCR primer; (ii) a PCR stopper to prevent PCR read-through of theprobe element; (iii) a specific probe sequence; and (iv) a fluorescencedetection system containing at least one fluorophore and quencher. AfterPCR extension of the Scorpion primer, the resultant amplicon contains asequence that is complementary to the probe, which is renderedsingle-stranded during the denaturation stage of each PCR cycle. Oncooling, the probe is free to bind to this complementary sequence,producing an increase in fluorescence, as the quencher is no longer inthe vicinity of the fluorophore. The PCR stopper prevents undesirableread-through of the probe by Taq DNA polymerase.

Invader assays (Third Wave Technologies, Madison, Wis.) are usedparticularly for SNP genotyping and utilize an oligonucleotide,designated the signal probe that is complementary to the target nucleicacid (DNA or RNA) or polymorphism site. A second oligonucleotide,designated the Invader Oligo, contains the same 5′ nucleotide sequence,but the 3′ nucleotide sequence contains a nucleotide polymorphism. TheInvader Oligo interferes with the binding of the signal probe to thetarget nucleic acid such that the 5′ end of the signal probe forms a“flap” at the nucleotide containing the polymorphism. This complex isrecognized by a structure specific endonuclease, called the Cleavaseenzyme. Cleavase cleaves the 5′ flap of the nucleotides. The releasedflap binds with a third probe bearing FRET labels, thereby forminganother duplex structure recognized by the Cleavase enzyme. This timethe Cleavase enzyme cleaves a fluorophore away from a quencher andproduces a fluorescent signal. For SNP genotyping, the signal probe willbe designed to hybridize with either the reference (wild type) allele orthe variant (mutant) allele. Unlike PCR, there is a linear amplificationof signal with no amplification of the nucleic acid. Further detailssufficient to guide one of ordinary skill in the art are provided by,for example, Neri, B. P., et al., Advances in Nucleic Acid and ProteinAnalysis 3826:117-125, 2000) and U.S. Pat. No. 6,706,471.

A variety of multiplex amplification systems can be used in conjunctionwith the present invention. In one type, several different targets canbe detected simultaneously by using multiple differently labeled probeseach of which is designed to hybridize only to a particular target.Since each probe has a different label, binding to each target to bedetected based on the fluorescence signals. By judicious choice of thedifferent labels that are utilized, analyses can be conducted in whichthe different labels are excited and/or detected at differentwavelengths in a single reaction. See, e.g., Fluorescence Spectroscopy(Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al.,Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York,(1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules,2nd ed., Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Genotyping

Many diseases linked to genome modifications, either of the hostorganism or of infectious organisms, are the consequence of a change ina small number of nucleotides, frequently involving a change in a singlenucleotide. Such single nucleotide changes are referred to as singlenucleotide polymorphisms or simply SNPs, and the site at which the SNPoccurs is typically referred to as a polymorphic site. The devicesdescribed herein can be utilized to determine the identify of anucleotide present at such polymorphic sites. As an extension of thiscapability, the devices can be utilized in genotyping analyses.Genotyping involves the determination of whether a diploid organism(i.e., an organism with two copies of each gene) contains two copies ofa reference allele (a reference-type homozygote), one copy each of thereference and a variant allele (i.e., a heterozygote), or contains twocopies of the variant allele (i.e., a variant-type homozygote). Whenconducting a genotyping analysis, the methods of the invention can beutilized to interrogate a single variant site. However, as describedfurther below in the section on multiplexing, the methods can also beused to determine the genotype of an individual in many different DNAloci, either on the same gene, different genes or combinations thereof.

Genotyping analyses can be conducted using a variety of differentapproaches. In these methods, it is generally sufficient to obtain a“yes” or “no” result, i.e., detection need only be able to answer thequestion whether a given allele is present. Thus, analyses can beconducted only with the primers or nucleotides necessary to detect thepresence of one allele potentially at a polymorphic site. However, moretypically, primers and nucleotides to detect the presence of each allelepotentially at the polymorphic site are included.

Single Base Pair Extension (SBPE) reactions are one techniquespecifically developed for conducting genotyping analyses. Although anumber of SPBE assays have been developed, the general approach is quitesimilar. Typically, these assays involve hybridizing a primer that iscomplementary to a target nucleic acid such that the 3′ end of theprimer is immediately 5′ of the variant site or is adjacent thereto.Extension is conducted in the presence of one or more labelednon-extendible nucleotides that are complementary to the nucleotide(s)that occupy the variant site and a polymerase. The non-extendiblenucleotide is a nucleotide analog that prevents further extension by thepolymerase once incorporated into the primer. If the addednon-extendible nucleotide(s) is(are) complementary to the nucleotide atthe variant site, then a labeled non-extendible nucleotide isincorporated onto the 3′ end of the primer to generate a labeledextension product. Hence, extended primers provide an indication ofwhich nucleotide is present at the variant site of a target nucleicacid. Such methods and related methods are discussed, for example, inU.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and5,710,028; and in WO 92/16657.

Genotyping analyses can also be conducted using quantitative PCRmethods. In this case, differentially labeled probes complementary toeach of the allelic forms are included as reagents, together withprimers, nucleotides and polymerase. However, reactions can be conductedwith only a single probe, although this can create ambiguity as towhether lack of signal is due to absence of a particular allele orsimply a failed reaction. For the typical biallelic case in which twoalleles are possible for a polymorphic site, two differentially labeledprobes, each perfectly complementary to one of the alleles are usuallyincluded in the reagent mixture, together with amplification primers,nucleotides and polymerase. Sample containing the target DNA isintroduced into the reaction site. If the allele to which a probe iscomplementary is present in the target DNA, then amplification occurs,thereby resulting in a detectable signal as described in the detectionabove. Based upon which of the differential signal is obtained, theidentity of the nucleotide at the polymorphic site can be determined. Ifboth signals are detected, then both alleles are present. Thermocyclingduring the reaction is performed as described in the temperature controlsection supra.

Gene Expression

Gene expression analysis involves determining the level at which one ormore genes is expressed in a particular cell. The determination can bequalitative, but generally is quantitative. In a differential geneexpression analysis, the levels of the gene(s) in one cell (e.g., a testcell) are compared to the expression levels of the same genes in anothercell (control cell). A wide variety of such comparisons can be made.Examples include, but are not limited to, a comparison between healthyand diseased cells, between cells from an individual treated with onedrug and cells from another untreated individual, between cells exposedto a particular toxicant and cells not exposed, and so on. Genes whoseexpression levels vary between the test and control cells can serve asmarkers and/or targets for therapy. For example, if a certain group ofgenes is found to be up-regulated in diseased cells rather than healthycells, such genes can serve as markers of the disease and canpotentially be utilized as the basis for diagnostic tests. These genescould also be targets. A strategy for treating the disease might includeprocedures that result in a reduction of expression of the up-regulatedgenes.

The design of the devices enables them to be utilized in combinationwith a number of different heating systems. Thus, the devices are usefulin conducting diverse analyses that require temperature control.Additionally, those microfluidic devices adapted for use in heatingapplications can incorporate a further design feature to minimizeevaporation of sample from the reaction sites. Devices of this type ingeneral include a number of guard channels and/or reservoirs or chambersformed within the elastomeric device through which water can be flowedto increase the water vapor pressure within the elastomeric materialfrom which the device is formed, thereby reducing evaporation of samplematerial from the reaction sites.

In another embodiment, a temperature cycling device may be used tocontrol the temperature of the microfluidic devices. Preferably, themicrofluidic device would be adapted to make thermal contact with themicrofluidic device. Where the microfluidic device is supported by asubstrate material, such as a glass slide or the bottom of a carrierplate, such as a plastic carrier, a window may be formed in a region ofthe carrier or slide such that the microfluidic device, preferably adevice having an elastomeric block, may directly contact theheating/cooling block of the temperature cycling device. In a preferredembodiment, the heating/cooling block has grooves therein incommunication with a vacuum source for applying a suction force to themicrofluidic device, preferably a portion adjacent to where thereactions are taking place. Alternatively, a rigid thermally conductiveplate may be bonded to the microfluidic device that then mates with theheating and cooling block for efficient thermal conduction resulting.

The array format of certain of the devices means the devices can achievehigh throughput. Collectively, the high throughput and temperaturecontrol capabilities make the devices useful for performing largenumbers of nucleic acid amplifications (e.g., polymerase chain reaction(PCR)). Such reactions will be discussed at length herein asillustrative of the utility of the devices, especially of their use inany reaction requiring temperature control. However, it should beunderstood that the devices are not limited to these particularapplications. The devices can be utilized in a wide variety of othertypes of analyses or reactions.

If the device is to be utilized in temperature control reactions (e.g.,thermocycling reactions), then, as described in greater detail infra,the elastomeric device is typically fixed to a support (e.g., a glassslide). The resulting structure can then be placed on a temperaturecontrol plate, for example, to control the temperature at the variousreaction sites. In the case of thermocycling reactions, the device canbe placed on any of a number of thermocycling plates.

Because the devices are made of elastomeric materials that arerelatively optically transparent, reactions can be readily monitoredusing a variety of different detection systems at essentially anylocation on the microfluidic device. Most typically, however, detectionoccurs at the reaction site itself (e.g., within a region that includesan intersection of flow channels or at the blind end of a flow channel).The fact that the device is manufactured from substantially transparentmaterials also means that certain detection systems can be utilized withthe current devices that are not usable with traditional silicon-basedmicrofluidic devices. Detection can be achieved using detectors that areincorporated into the device or that are separate from the device butaligned with the region of the device to be detected.

Utilizing systems and methods provided according to embodiments of thepresent invention, throughput increases are provided over 384 wellsystems. As an example, throughput increases of a factor of 4, 6, 12,and 24 and greater are provided in some embodiments. These throughputincreases are provided while reducing the logistical friction ofoperations. Moreover the systems and methods of embodiments of thepresent invention enable multiple assays for multiple samples. Forexample, in a specific embodiment 24 samples and 24 assays are utilizedto provide a total of 576 data points. In another embodiment, 32 samplesand 32 assays are utilized to provide a total of 1024 data points. Inanother embodiment, 48 samples and 48 assays are utilized to provide2304 data points. In another embodiment, 96 samples and 48 assays areutilized to provide 4608 data points. In another embodiment, 96 samplesand 96 assays are utilized to provide a total of 9,216 data points. In aparticular example, the 96 assays are components of a TaqMan 5′ NucleaseAssay. See, e.g., U.S. Pat. Nos. 5,538,848, 5,723,591, 5,876,930,6,030,787, 6,258,569, and 5,804,375, each of which is hereinincorporated by reference.

Depending on the geometry of the particular microfluidic device and thesize of the microfluidic device and the arrangement of the fluidcommunication paths and processing site, embodiments of the presentinvention provide for a range of reaction chamber. In some embodiments,the methods and systems of the present invention are utilized withchamber densities ranging from about 100 chambers per cm² to about 1million chambers per cm². Merely by way of example, microfluidic deviceswith chamber densities of 250, 1,000, 2,500, 10,000, 25,000, 100,000,and 250,000 chambers per cm² are utilized according to embodiments ofthe present invention. In some embodiments, chamber densities in excessof 1,000,000 chambers per cm² are utilized, although this is notrequired by the present invention.

Operating microfluidic devices with such small reaction volumes reducesreagent usage as well as sample usage. Moreover, some embodiments of thepresent invention provide methods and systems adapted to performreal-time detection, when used in combination with real-timequantitative PCR. Utilizing these systems and methods, six orders oflinear dynamic range are provided for some applications as well asquantitative resolution high enough to allow for the detection ofsub-nanoMolar fluorophore concentrations in 10 nanoliter volumes. One ofordinary skill in the art would recognize many variations,modifications, and alternatives.

Reactions may be designed to produce a detectable signal (indication)including fluorescent indications, but luminescent indications,including chemiluminescent, electroluminescent, electrochemiluminescent,and phospholuminescent, bioluminescent, and other luminescent processes,or any other processing involving any other type of indications that maybe detected using a detection device. As will be evident to one of skillin the art, methods and systems operable in the detection and analysisof these fluorescent and luminescent indications are transferable fromone indication to another. Additionally, although some embodiments ofthe present invention utilize spectral filters as optical elements, thisis not required by the present invention. Some fluorescent andluminescent applications do not utilize spectral filters in the opticalexcitation path, the optical emission path, or both. As describedherein, other embodiments utilize spectral filters. One of skill in theart will appreciate the differences associated with particularapplications.

In some embodiments, a variety of devices and methods for conductingmicrofluidic analyses are utilized herein, including devices that can beutilized to conduct thermal cycling reactions such as nucleic acidamplification reactions. The devices differ from conventionalmicrofluidic devices in that they include elastomeric components; insome instances, much or all of the device is composed of elastomericmaterial. For example, amplification reactions can be linearamplifications, (amplifications with a single primer), as well asexponential amplifications (i.e., amplifications conducted with aforward and reverse primer set).

IX. EXAMPLES A. Example 1 Fabrication of a Matrix Reaction Array

A 32×32 elastomeric microfluidic matrix reaction array with slug mixingwas constructed and mixing efficiency was compared to a conventional32×32 elastomeric microfluidic matrix reaction array constructed with asample/reaction chamber adjacent to a reagent chamber and separated byan interface valve.

The matrix reaction array was constructed with each reaction cellcomprising a central 50 nL reaction chamber (400) and a 5 nL slugchannel (250) as shown in FIG. 2. The unit reaction cell of FIG. 2 wasrepeated to prepare a 32×32 matrix fluidic circuit. Using multilayersoft lithography, a first pour layer was patterned with SU8 photoresistto form a mold and then cast with PDMS. The features of the pour layerincluded a 350 um tall reaction chamber and a 30 um tall slug channel.The other feature, a connecting channel (300) was constructed with aheight of 10 um. A second spin layer was prepared with 15 um tallfeatures for sample input channel (220) and control channel 1 (260) andcontrol channel 2 (270). The second layer was laser punched to form thesample-to-slug via (240). The first pour layer was aligned and bonded tothe second spin layer and the bonded assembly then removed from theresist pattern. The bonded assembly comprising layers 1 and 2 was thenbonded to a thin spun base layer to complete the elastomeric assembly.

FIG. 1 depicts a representative portion of the 32×32 matrix fluidiccircuit. The matrix circuit was divided by column (e.g., C1, C2, C3, C4)and rows (e.g. R1, R2, R3, R4). The slug channels 250 of cells in aparticular column, such as column C4, are in fluidic communication viaconnecting channels (300). Connecting channel 300 constitutes a fillinlet for the slug channel. A valve defined by a deflectable membrane ofcontrol channel 2 (270) can fluidically isolate the slug channels of theindividual unit cells of a given column. The isolation of the slugchannels from each other is accomplished by pressurization of controlchannel 2 causing the deflectable membrane portions to deflect into theconnecting channels and sealing off flow through the connectingchannels. The slug channels of a given column are thereforeinterconnected in their native state and capable of being isolated uponactuation of control channel 2. A similar arrangement of interconnectsexists for the rows of the matrix device. A common bus line, sampleinput channel (220), exists in the second layer of the device. This busline provides a common sample input for all of the unit reaction cellsof a particular row of the device. The sample input channel is connectedto the individual slug channels through the sample-to-slug fluidcommunication via connecting the sample input channel in the secondlayer to the first end of the slug channel in the first layer. Thesecond end of the slug channel connects directly to the reaction chamber(400). The configuration of the slug channel allows the first end to befluidically isolated from the sample-to-slug fluid communication via andthe second end to be isolated from the reaction chamber with theactuation the valves formed from control channel 1. The pressurizationof control channel 1, which resides in the second layer, causes thedeflection of a membrane in the top of the control channel to deflectinto slug channel near the first end and the second end to fluidicallyisolate the ends of the slug channel.

B. Example 2 Demonstration of the Filling and Mixing of a 32×32 MatrixReaction Array

Operation of the matrix reaction array was performed by the followingsteps. In this operation a yellow food dye was used as a surrogate forthe first solution (reagent) and a blue food dye was used as a surrogatefor the second solution (sample). Flow and mixing could be monitoredbased on the colored solutions. FIG. 3 illustrates the result of theoperation and is based on photomicrographs taken. As noted above, FIG. 3is illustrative only. The loading time in this experiment was from 10-20minutes and, under the conditions of the demonstration, diffusiondominated mixing occurred within the reagent channel before thesolutions entered the reaction chamber.

Control channel 1 (260) was pressurized to close the valves thatfluidically isolate the ends of the slug channel (FIG. 3A). A yellowfood dye was introduced under pressure through the connecting channels(300) and the slug channels were blind-filled (FIG. 3B). This stepsimulates the filling of the slug channel with reagent. Following thefilling of the slug channels, control channel 2 (270) is pressurized toactuate the valves that close off the connecting channels (300) andthereby isolate the individual slug channels from the other slugchannels in the columns (FIG. 3C). Although all columns in the reactionarray and, accordingly, all slug channels, are filled with the sameyellow food dye in this example, there is no interconnection between theconnecting channels and the slug channels of the individual columns.Following the blind filling of the slug channels and their isolation, ablue food dye was introduced under pressure into all sample inputchannels (220) (FIG. 3D). The control channels 1 (260) were thendepressurized to open the interface valves that were previously closedto isolate the ends of the slug channels (FIG. 3E). The blue food dyethat represents the sample in this Example, enters the slug channel atthe first end and pushes the reagent into the reaction chamber (FIG.3F). This resulted in a highly mixed, loaded reaction chamber (400)containing the 5 nL of yellow food dye reagent surrogate and 45 nL ofblue food dye sample surrogate (50 nL total reaction chamber volume)(FIG. 3G). Finally, in this demonstration, control channels 1 arepressurized which results in the closure of the interface valves (FIG.3H). Although all rows in the reaction array and, accordingly, allsample input channels, are filled with the same blue food dye in thisexample, there is no interconnection between the sample input channelsof the individual rows and different samples can be introduced into theindividual rows. In the configuration of this Example, 32 separatesamples can be simultaneously mixed and loaded into reaction chamberswith 32 separate reagents for 1024 individual experiments.

C. Example 3 Fabrication and Operation of FCS Device (Exemplar 5-Type)

In this example, reaction cells are fabricated in 100 nL (FIG. 5)volumes by multilayer soft lithography. A reaction chamber 400 isprepared on a first spin layer of varying thicknesses. For a 100 nLreaction chamber, a 100 um recess is patterned with SU8 photoresist anda first polydimethylsiloxane (PDMS) elastomeric layer is prepared byspin coating the resist pattern to define a 100 nL reaction chamber(400) with an open side and a closed side. Once the first layer iscured, a via (280) is laser punched through the closed side of thereaction chamber. A second elastomeric layer is prepared by spin coatinga resist pattern to define a 10 um rounded reagent slug channel (250) influidic communication with a 30 um reagent input flow channel (230). Thechannels are formed as recesses in the second layer. The reagent inputflow channel is formed as a bus channel to connect with additionalreagent slug flow channels when forming multiple reaction cells. Whenthe second layer is cured, via (240) is laser punched through theceiling of one end of the reagent slug flow channel. A third elastomericlayer is prepared as a pour layer over a photoresist mold. Thephotoresist pattern defines recesses for a first 28 um control channel(260) and a second 28 um control channel (270). The control channelshave a widened recess area that is intended to overlie the ceilingmembranes of the flow channels for which they are intended to control.When the control channel is sufficiently pressurized, the ceilingmembrane of the flow channel that is beneath the widened control channelrecess area will be deflected into the flow channel beneath it thussealing off the flow channel. When pressure is reduced or removed, theceiling membrane of the flow channel will deflect upward to reopen theflow channel. A narrow portion of the control channel carries thecontrol fluid for pressurizing the control channel. By selecting propercontrol channel geometries and flow channel ceiling membranethicknesses, the narrow portion of the control channel overlies portionsof flow channels but does not deflect the flow channels ceiling membraneinto the flow channel at these intersections upon pressurization of thecontrol channel. Also defined in the third elastomeric layer is recessfor a sample inlet channel (220). Once the third elastomeric layer iscured, it is removed from the photoresist mold. The layers are thenaligned and assembled—first by assembling the “third” and “second”layers, and then by assembling the “third/second” layer with the“first”. The elastomeric layers are bonded together by first plasmatreating the surfaces of the layers and then contacting the layers. Thelayers are aligned such that: 1) the first layer is placed with thereaction chamber opening downward; 2) second elastomeric layer isaligned on top of the first layer so that the reagent slug channelrecess (250) is in fluid communication with the slug to reaction chambervia (280); and 3) the third elastomeric layer is aligned so that therecess defining the sample input channel (240) is in fluid communicationwith the sample to slug via (240). The third elastomeric layer is alsoaligned so that the recesses that define control channel 1 (260) with awidened control channel recess area overlies two ends of the reagentslug flow channel. The widened control channel recess area of controlchannel 2 (270) is aligned so that it overlies a portion of the reagentslug flow channel that interfaces to the reagent input flow channel(230). The assembled elastomeric layers form a microfluidic reactioncell that is bonded to a silicon base layer. In this example, the baselayer is a solid monolithic slab of silicon that seals the open end ofthe reaction chamber and also functions as a heat transfer surface fortemperature control of reactions such as polymerase chain reaction(PCR).

Reaction chamber volumes of 10 nl and 1.5 nl having the same generaldesign as described above were also prepared, with 60 um recess in thefirst elastomeric layer that defines the reaction chamber depth and withreduced length and width dimensions.

To operate the carry-slug reaction cell, control channel 1 [260] ispressurized to deflect the elastomeric membrane valve and close the slugreagent flow channel at its interface with the sample to slug via [240]and the slug to reaction chamber via [280] The reagent input flowchannel [230] is pressurized and the entire slug reagent flow channel[250] is blind filled with the desired reagent. Simultaneously, thesample input flow channel is also pressurized and the entire sample flowchannel is blind filled up to the valve delineated by control channel 1.Control channel 2 is then pressurized to deflect the elastomericmembrane valve that closes the slug reagent flow channel near itsinterface with the reagent input flow channel. The sample input flowchannel is then re-pressurized and control channel 1 is depressurized toopen the slug reagent flow channel at its via connections. The contentsof the slug reagent flow channel are then transferred into the reactionchamber through blind filling and under pressure from the sample inputflow channel. The volume of the reaction chamber is in excess of thevolume of the slug reagent flow channel which allows for sample tocontinue flowing from the sample input flow channel and the slug reagentflow channel and to fill up the reaction chamber in the amount that isthe difference of the reaction chamber volume and the slug reagent flowchannel volume. Control channel 1 is repressurized to close off thereaction chamber. The reagent and sample are retained in a mixedsolution in the reaction chamber and the reaction is allowed to proceed.

D. Example 4 Flow Channel Provided with Multiple Mixing Segments

FIG. 7C is a representation of a flow channel that provides for multipleslug mixing segments. Flow channel (250, comprising segments 250 a, 250b, 250 c and 250 d) has a first end gated by valve V8 and a second endgated by valve V1 that opens into reaction chamber (400). Along thelength of the flow channel are multiple junction inlets: 303, 302, 301,and 300, gated by valves V9, V7, V5, and V3, respectively. Each slugmixing segment is defined by a valve pair that brackets the inletjunction. A first slug mixing segment 250 d is defined by the segment ofthe flow channel defined by inlet channel 303 and valves V8 and V6. Asecond slug mixing segment 250 c is defined by inlet channel 302 andvalves V6 and V4. A third slug mixing segment 250 c is defined by inletchannel 301 and valves V4 and V2. A fourth slug mixing segment 250 a isdefined by inlet channel 300 and valves V2 and V1. In this type ofarrangement, multiple solutions may be introduced into the flow channelby blind filing against the valves that define their respectivesegments. Their inlet junction valves are then closed, the segmentvalves are opened and the slugs are pushed into the reaction chamber byflow of a solution through the flow channels 220 and 290 to yield a wellmixed solution. It is not necessary that all segments of the flowchannel are filled with a solution before the carry-on mixing takesplace. This gives flexibility in what reagents are used in a particularreaction.

The reaction chamber 400 may have has an optional outlet channel gatedby a valve.

E. Example 5 FCS Unit Cell and High Density Array

A high density nanofluidic chip with a FCS design unit cell wasconstructed. The unit cell was designed for a 10:1 mixing ratio and a6.75 μL mixing/reaction chamber. The nanofluidic chip was constructedfrom three elastomeric layers: A first layer containing control andsample bus channels with a layer thickness of approximately 35 μm; asecond layer containing reagent bus channels, reagent slug channels, andmixing/reaction chambers with a layer thickness of approximately 4 mm,and a third base layer with a thickness of approximately 100 μm andcontaining vias for fluidic communication of the nanofluidic device withthe external world. As the mixing (and potential reaction) of the sampleand the reagent take place in the second layer, vias are formed betweenthe first and second layers to allow transfer of sample from the samplebus (sample input channel) in the first layer to the reagent slugchannel of the second layer and, ultimately, allow the flow of thesample into the mixing/reaction chamber. The chip architecture wasdesigned as matrix of 48 rows and 48 columns for a total of 2304 unitcells for mixing and potential reactions. In this design, 48 independentsamples can be individually mixed with 48 independent reagents. For a48×48 matrix design, the features of the unit cell must be replicatedacross the rows and down the columns of the matrix. For each cell of thematrix of the present example to be independently addressable, each cellrequires a unit cell via, or other connection between the first andsecond layers. In this example, the entire matrix of 2304 unit cells iscontained within a 30 mm×30 mm area of the nanofluidic chip.

FIG. 4 is a diagram of a portion of the nanofluidic chip of the presentexample. The figure shows four of the unit cells arranged in a matrix(two rows and two columns) that are part of the larger 48×48 matrix. Theunit cell comprises a mixing or reaction chamber (400), a reagent slugchannel (250), a reagent input channel (230), a sample input channel(220), a first control channel (260), and a second control channel(270). In this example, a unit cell via (240) connects the sample inputchannel to the reagent slug channel through two layers of thenanofluidic chip. A mold was patterned with photoresist that defines afirst control channel (260), a second control channel (270) and sampleinput channel (220) layer. The narrowest portions of the mold for bothtypes of channels were 15 μm (height) by 35 μm (width). Portions of thefirst control channel were widened (65 μm) and portion of the secondcontrol channel were widened (70 μm) at areas where the valve portionswere defined. A layer of polydimethylsiloxane (PDMS) was spin coatedover the resist pattern mold to produce the first layer. A second layerwas prepared by pouring PDMS over a resist pattern that defined a 10 μmrounded reagent input channel (230) in communication with a 10 μm×85 μm(height×width) reagent slug channel (250). For the resist defining theportion of the reagent input channel that overlays the valve portion ofthe second control channel (270), the width was 85 μm wide. The resistthat defines the portion of reagent input channel 230 that interfaceswith the reagent slug channel was defined with a height of 30 μm. Theresist forming the mold for mixing/reaction chamber 400 was defined withthe dimensions of 270 μm×200 μm×125 μm (height×width×length). The secondlayer was poured to a thickness of approximately 4 mm, allowed to cure,and removed from the resist pattern. The mold-facing-surface of thesecond layer (bottom) and the non-mold-facing surface of the first layer(top) were plasma treated and the second layer was aligned over thefirst layer so that the unit cell vias in the first layer are in fluidiccommunication with the reagent slug channels of the unit cells in thesecond layer. The second layer was seated on top of the first layer(which remained on the resist pattern mold) and the compositionstructure was baked in order to bond the layers together. A third, 100μm thick layer of PDMS was spun on a support and cured. Vias were laserpunched in the third layer to provide fluidic communication from thebottom of the device to the reagent bus lines, the sample bus lines, andthe first and second control channels. With this configuration, samplesand reagents can be loaded from the bottom periphery of the completednanofluidic device and fluidic pressure can be introduced into thecontrol channels from the bottom of the device. The top surface of thethird layer was plasma treated. The first and second layer compositestructure was peeled from the first layer resist pattern mold and themold-facing-side was plasma treated. The bottom of the compositestructure was aligned and applied to the third layer to form thecompleted nanofluidic device. The device was baked to finish bonding thefirst and second layer composite to the third layer. The device was thenremoved from the third layer support. The completed device wasapproximately 43 mm×43 mm×4 mm. The 48 rows and 48 columns of unit cellswere contained with an area of the device that was approximately 30mm×30 mm. The bottom of the device (the surface comprised of the thirdlayer) was coated with an adhesive and bonded to silicon wafer ofapproximate 33 mm×33 mm×525 μm (width×length×height) that acts as athermal transfer device for controlling the temperature of the samplesand reagents and mixed sample/reagent solutions that are loaded withinthe nanofluidic device.

The nanofluidic device of this example is of a matrix configuration thatallows for the independent mixing of 48 individual samples (firstsolutions) with 48 individual reagents (second solutions). Thereforethere are 48 individual sample input channels and 48 individual reagentinput channels. There is a first control channel and a second controlchannel for each unit cell that can be configured to be independentlyaddressable but, in this example, all first control channels (and thevalves that they define) operate together and all second controlchannels (and the valves that they define) operate together.

To operate the device, the first control channel is pressurized todeflect the valve and close the reagent slug channel at both itsinterface with the unit cell via and its interface with themixing/reaction chamber. The reagent input channel is pressurized andthe reagent slug channel is blind filled with the desired reagent. Thesample input channel is pressurized and the sample input channel isblind filled up to the valve delineated by the first control channel.The second control channel is then pressurized to isolate the reagentslug channel. The first control channel is depressurized to place thesample input channel, reagent slug channel, and mixing/reaction chamberin serial fluidic communication. The flow of sample pushes the contentsof the reagent slug channel into the mixing/reaction chamber throughblind filling and under pressure from the sample input flow channel. Thevolume of the reaction chamber is in excess of the volume of the reagentslug channel which allows for sample to continue flowing from the sampleinput channel through the reagent slug channel and to fill up themixing/reaction chamber in the amount that is the difference of themixing/reaction chamber volume and the reagent slug channel volume. Thefirst control channel is then pressurized to close off the reactionchamber. The reagent and sample are retained in a mixed solution in thereaction chamber and the reaction is allowed to proceed. For the deviceof this example, the thermal transfer device was coupled to a thermalcontroller for conducting polymerase chain reactions within themixing/reaction chambers.

F. Example 6 Microfluidic Check Valves

This example describes a microfluidic check valve that may be used invarious FCS devices. The check valve is comprised two stacked chambersthat are separated by a pore-containing membrane. The membrane iscomposed of an elastomeric material and can be configured in normallyopen or normally closed state. The normally open check valve, whichgenerally will be used in the FCS devices, can be implemented so thatthe degree of back pressure necessary to close the valve can be set.Both the normally open and the normally closed version can be readilyproduced by multilayer soft lithographic techniques. Further discussionof this valve is found in PCT application PCT/US07/80489, filed 4 Oct.2007, the entire content of which is enclosed herein in its entirety.

FIG. 10 shows an exemplary valve. An upper layer (507) defines an outletchamber (501) that is in fluidic communication with and outlet channel(506). The outlet chamber has a height, D, and a chamber width, C. Theupper layer is adhered to, pressed onto, or bonded to the membrane (503)with its flow channel (504) opening into the outlet chamber. Themembrane has a thickness, F, and a flow channel width (or diameter), E.The membrane layer is adhered to, pressed onto, bonded to, or integralwith the bottom layer (508) which defines the input chamber (502) andthe input flow channel (505). The input chamber has a width (ordiameter), A, and a height, B. The layer 508 is adhered to, pressedonto, or bonded to a substrate (either hard or elastomeric) (509) thatforms the inlet channel (505).

In this valve, the footprint of the inlet chamber has a shortestinternal width, A, and the inlet chamber has a height, B, the footprintof the outlet chamber has a shortest internal width, C, and the outletchamber has a height, D. In an embodiment, the membrane channel has awidth, E, and a membrane thickness, F. The check valves of the inventionwill typically have a ratio of C to A is greater than or equal to about1.2, a ratio of D to B is greater than or equal to about 1.4, and aratio of A to E is greater than or equal to about 1.9. In furtherembodiments, the ratio of C to A is equal to or less than about 1.5,equal to or less than about 1.75, equal to or less than about 2, equalto or less that about 2.5, equal to or less than about 3, or greaterthan 3. The ratio of D to B can be equal to about 1.6 or less, equal toor less than about 1.8, equal to or less than about 2, equal to or lessthan about 2.5, or equal to or less than about 3, or greater than 3. Theratio of A to E can be equal to or less than about 2.2, equal to or lessthan about 2.5, equal to or less than about 2.8, equal to or less thanabout 3, or greater than 3. The membrane thickness, F, can be from about2 to about 100 um, preferably from about 2 to about 75 um, preferablyfrom about 2 to about 50 um, more preferably from about 2 to about 25um. In some embodiments, it is preferred that F is less than about 25um. In some embodiments it is preferred that F is equal to or less thanabout 10 um. In other embodiments, it is preferred that F is equal to orless than 5 um in thickness. The membrane (503) should have a Young'smodulus of about 100 MPA (megapascals) or less. In other embodiments,the Young's modulus of the membrane is about 75 MPA or less, about 50MPa or less, about 25 MPa or less, about 10 MPa or less, about 8 MPa orless, about 5 MPa or less, or about 2 MPa or less.

The check valve may be used in a device comprising, for example, aninlet channel segment, a check valve, and an outlet channel segmentwherein, in the absence of outlet channel flow restrictions, an inletchannel pressure of less than 5 psi (pounds per square inch) is requiredto produce flow to the outlet channel and wherein substantially no flowoccurs from the outlet channel to the inlet channel when an outletpressure exceeds the inlet channel pressure by about 3 psi. In a furtherembodiment, the check valve will allow flow to occur from the inletchannel to the outlet channel with an inlet channel pressure of lessthan 3 psi, 2 psi, 1 psi, 0.5 psi or 0.2 PSI. The initial inlet pressurerequired to open the check valve will, in some cases, exceed thepressure required to open the check valve in subsequent opening. Theopening pressures recited above represent the average opening pressuresof 10 repeated openings and closings within a 30 minutes period. In anembodiment, the check valve will close when the pressure in the outletchannel exceeds the pressure in the inlet channel by 2 psi, 1 psi, 0.5psi, 0.25 psi, 0.1 psi, or 0.05 psi. In a further embodiment, the checkvalve will close when the pressure in the outlet channel exceeds thepressure in the inlet channel by 0.005 psi.

The check valves are further characterized by a very low dead volume.The check valves my have a dead volume of 100 nL (nanoliters) or less,50 nL or less, 25 nL or less, 15 nL or less, 10 nL, or less, 5 nL orless, 4 nL or less, 2.5 nL or less, or, in a further embodiment, about 1nL.

FIG. 11 depicts a fabrication process for a microfluidic check valve. Inthis Example, the microfluidic check valve utilized two elastomericlayers bonded to each other and attached to a substrate. The substratecould be elastomeric or made of a rigid material. Column A shows thefabrication of the bottom (inlet) chamber (201) and the membrane (203)with a pore (flow channel) (204). Column B shows the fabrication of thetop (outlet) chamber (201). The fabrication of the bottom chamber beginswith a photoresist lithographic process that produces a mold (220 and225) defining a chamber with a thin membrane roof and an inlet channel(Column A (1)). An uncured, liquid elastomeric compound such aspolydimethylsiloxane (PDMS) is spin-layered onto the mold to evenlydistribute the elastomeric liquid on the surface of the mold (Column A(2)) and the elastomeric liquid is cured or is allowed to cure. Theelastomer is pealed off the mold, and the mold (Column A (3)) and a hole(204) is punched in the membrane roof (203) of the bottom chamber(Column A (4)). The top chamber is fabricated by a similar process, i.e.by spin-layering an elastomeric liquid on lithographically produced spinmold (230 and 225) that defines the top chamber (201) and the outletflow channel (206), curing the spun elastomeric layer (207), andremoving the cured elastomer with the molded features (Column B (1′),(2′) and (3′)). The two elastomeric layers are assembled by bonding thelayer defining the top chamber onto the elastomeric layer defining thebottom chamber, membrane, and pore. The layers are aligned such that thecavity defining the top chamber is aligned with the membrane roof of thebottom chamber so that sufficient membrane remains unbonded and pliableto allow actuation (5). Assembly of the microfluidic check valve iscompleted by attachment of the bonded elastomeric structure to asubstrate (carrier) by gluing or bonding the side of the elastomericstructure with the cavity defining the bottom chamber to the substrate(209) so that connections to liquid flow channels are obtained (6).Alternate embodiments are possible including a flow channel defined inthe carrier to make direct connection to the bottom chamber ofconnection to a recess in the elastomeric structure that defines aninput flow channel leading to the bottom chamber.

G: Example 7 Single Nucleotide Polymorphism (SNP) Analysis

This example describes a comparison of SNP analysis using real-timequantitative PCR (rt-qPCR) carried out using an Exemplar 2-Type FCSdevice with a 48×48 unit cell array (M48CS2), as described in Example 5,and a prior art device with a 48×48 unit cell array (M48; described inUS Pat. Pub. US 2005/0145496, FIG. 21; Fluidigm Corp., S. San FranciscoCalif.).

45 different TaqMan assay sets specific for different single nucleotidepolymorphisms (SNPs) in salmon DNA were used in this study. Primers andprobes were used at final concentrations of 9 uM and 2 uM respectively.The TaqMan probes used were dual-labeled, dark hole quenchedoligonucleotides. Forty-six different salmon DNA samples obtained fromthe Alaska Department of Fish and Game were tested for the presence ofthe 45 SNPs, in parallel, on M48CS2 and M48 chips. Two negative samplecontrols and three negative reagent controls (buffer only) were also runon each chip.

Chips were primed, loaded and thermal cycled using standard PCRconditions, on the Fluidigm Dynamic Array System. Images were captured,and data was analyzed using a BioMark Analysis software package(Fluidigm Corp.) and genotyping call rates were then determined. Thecall rate obtained on either chip is shown in Table 1, below. Theaverage call rate on the M48CS2 chip was clearly better than that on theM48 chip (99.1% compared to only 93.6%).

TABLE 1 M48 M48Cs2 SNP02 One_ALDOB-135 95.3 97.8 SNP03 One_CO1 100.091.3 SNP04 One_CTGF-301 100.0 100.0 SNP05 One_Cytb_17 100.0 100.0 SNP06One_Cytb_26 100.0 100.0 SNP07 One_HGFA 93.0 100.0 SNP08 One_Hpal-43686.0 100.0 SNP09 One_Hpal-99 97.7 100.0 SNP10 One_IL8r-362 97.7 100.0SNP11 One_KPNA-422 97.7 100.0 SNP12 One_LEI-87 97.7 100.0 SNP13 One_Prl2100.0 100.0 SNP14 One_RAG1-103 100.0 100.0 SNP15 One_RAG3-93 100.0 100.0SNP16 One_RF-112 100.0 89.1 SNP17 One_RF-295 100.0 100.0 SNP18One_RH2op395 97.7 100.0 SNP19 One_U401-224 97.7 97.8 SNP20 One_U404-229100.0 100.0 SNP21 One_U502-167 100.0 100.0 SNP22 One_U503-170 100.0100.0 SNP23 One_U504-141 100.0 100.0 SNP24 One_U508-533 86.0 100.0 SNP25One_E2 95.3 97.8 SNP26 One_GHII-2461 100.0 100.0 SNP27 One_GPDH 100.097.8 SNP28 One_GPDH2 97.7 100.0 SNP29 One_GPH-414 95.3 100.0 SNP30One_hcs71-220 93.0 100.0 SNP31 One_MARCKS_241 97.7 100.0 SNP32One_MHC2_190 60.5 97.8 SNP33 One_MHC2_251 51.2 97.8 SNP34 One_Ots213-18197.7 97.8 SNP35 One_p53-576 97.7 100.0 SNP36 One_plns 65.1 100.0 SNP37One_serpin 86.0 100.0 SNP38 One_STC-410 74.4 100.0 SNP39 One_STR07 100.093.5 SNP40 One_Tf_ex10-750 97.7 100.0 SNP41 One_Tf_ex3-182 97.7 100.0SNP42 One_U301-92 90.7 100.0 SNP43 One_VIM-569 88.4 100.0 SNP44One_ZNF-61 83.7 100.0 SNP45 One_zP3b 100.0 100.0 SNP46 One_ACBP-79 97.7100.0 Average 93.6 99.1

Although the present invention has been described in detail withreference to specific embodiments, those of skill in the art willrecognize that modifications and improvements are within the scope andspirit of the invention, as set forth in the claims which follow. Allpublications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same. Theinvention having now been described by way of written description andexample, those of skill in the art will recognize that the invention canbe practiced in a variety of embodiments and that the foregoingdescription and examples are for purposes of illustration and notlimitation of the following claims.

1. A method for mixing solutions in a microfluidic device, wherein thedevice comprises a reaction chamber with a single flow channel connectedthereto, and wherein situated along the flow channel is a definedsegment bounded on the end proximal to the reaction chamber by a firstvalve, and bounded on the end distal to the reaction chamber with asecond valve, the method comprising: a) introducing a volume of a firstsolution into said segment by blind filling; b) flowing a volume of asecond solution through said segment, thereby displacing said firstsolution into the reaction chamber; and then c) flowing said volume ofsecond solution into the reaction chamber, whereby mixing of said firstand second solutions occurs in said reaction chamber.
 2. The method ofclaim 1, wherein at least one side of the flow channel has a materialthat is permeable to gases and not to liquid.
 3. The method of claim 2,wherein said material is an elastomeric material.
 4. The method of claim2, wherein introducing the first solution into the flow channel segmentcauses gas present in the flow channel to diffuse out through the gaspermeable material.
 5. The method of claim 1, wherein the first solutionto be delivered into the reaction chamber has a volume defined by theflow channel dimensions and the spacing of the first and second valvesalong the channel; and wherein after the first solution is introducedinto the segment, the second solution is introduced into an emptyportion of the flow channel by blind filling against the second valve.6. The method of claim 1, wherein the second solution is made to pushthe first solution through the flow channel into the reaction chamber bymaintaining the second solution under pressure, and opening the secondvalve.
 7. The method of claim 1, wherein the combined volumes of thefirst solution and the second solution equals the fluidic capacity ofthe reaction chamber.
 8. The method of claim 1, wherein the volume ofthe second solution in b) is at least twice the volume of the firstsolution in a).
 9. The method of claim 1, further comprising fluidicallyisolating said reaction chamber once the first and second solutions havebeen mixed.
 10. The method of claim 1, wherein the device comprises anarray of more than 25 rows and more than 25 columns of interconnectedunit cells.
 11. The method of claim 10, wherein the same first solutionis introduced into each unit cell in a column of the array, and the samesecond solution is introduced into each unit cell in a row of the array.12. The method of claim 11, wherein the first solution is introducedinto said column through a reagent bus line or reagent input channelfrom a reagent source, and the second solution is introduced into saidrow through a sample bus line from a sample source.
 13. The method ofclaim 11, wherein the sample bus line is situated in one layer of thedevice, and is connected to the defined segment of the flow channel inanother layer of the device by way of a vertical linking channel. 14.The method of claim 1, wherein the device is constructed from layers ofelastomeric materials.
 15. The method of claim 1, wherein the device hasa reaction chamber with a volume of 0.01 to 100 nanoliters.
 16. Themethod of claim 1, wherein the first solution comprises reagents foramplification of a nucleic acid and the second solution comprises anucleic acid sample.
 17. The method of claim 1, wherein the microfluidicdevice comprises an array of interconnected unit cells, wherein eachunit cell comprises: a) said single flow channel connected with: 1) saidreaction chamber, and 2) a first microfluidic bus line connected with asample source reservoir; b) a first valve situated in said flow channel;c) a second valve situated in said flow channel; d) a secondmicrofluidic flow path connected with: 1) said single flow channel at ajunction between said first and second valves, and 2) a secondmicrofluidic bus line connected with a reagent source reservoir; and e)a third valve situated 1) in said second microfluidic flow path, or 2)in said second microfluidic bus line positioned between the single flowchannel of said unit cell and the second microfluidic flow path of anadjacent unit cell.
 18. The method of claim 1, further comprisingclosing the first valve before the blind filling with the first solutionin a), and then opening the first valve before the flowing of the secondsolution in b).
 19. The method of claim 1, wherein the second valve is aone-way check valve.
 20. The method of claim 1, wherein the second valveis not a one-way check valve, and the method further comprises openingthe second valve before the flowing of the second solution in b). 21.The method of claim 9, wherein the reaction chamber is fluidicallyisolated by closing the first valve.
 22. The method of claim 9, whereinthe reaction chamber is fluidically isolated due to a one-way checkvalve that is proximal to the reaction chamber.
 23. A method for mixingsolutions in a microfluidic device according to claim 1, the methodcomprising: i) introducing a first solution into a segment of a flowchannel of a unit cell of said device, wherein said flow channel is influidic communication at a first end with a reaction chamber havingfluid capacity F; wherein a first position in said flow channel isproximal to a second position if the first position is fluidicallycloser to said reaction chamber said second position, and is distal tosaid second position if the first position is fluidically further fromthe reaction chamber than said second position; wherein said flowchannel segment is bounded by a first valve located in said flow channeland a second valve located in said flow channel, wherein said secondvalve is distal to said first valve; wherein said flow channel segmentis in fluidic communication through a first input junction with a sourceof said first solution, said first input junction being positionedbetween said first valve and said second valve; wherein said flowchannel segment is in fluidic communication through a second inputjunction with a source of said second solution, said second inputjunction being positioned distal to said second valve, wherein a thirdvalve is located between the second input junction and the source of thesection solution so that flow of said second solution into the flowchannel may be regulated; wherein the fluid capacity of said flowchannel segment is less than F; and wherein said first valve is closedwhen said first solution is introduced; wherein said second valve is inthe closed when said first solution is introduced, or is a check valvethat permits flow only toward the reaction chamber; wherein said thirdvalve is positioned such that when the first valve is closed, saidsecond valve is closed or is a check valve, and said third valve isclosed or is a check valve that permits flow only toward the reactionchamber, said first solution is retained in said segment; ii) if saidthird valve is not a check-valve, closing said third valve; iii)introducing said second solution under pressure into said segment, saidintroducing comprising: a) if said second valve is not a check valve: 1)flowing said second solution into a portion of said flow channel distalto the second valve before, after or concurrently with step (i) 2)opening said second valve 3) opening said first valve 4) flowing saidsecond solution into said segment of said flow channel, therebydisplacing said first solution into the reaction chamber, and 5) flowingsaid second solution into the reaction chamber; or b) if said secondvalve is a check valve: 1) flowing said second solution into the portionof said flow channel distal to the second valve 2) opening said firstvalve 3) flowing said second solution through the second valve, therebydisplacing said first solution into the reaction chamber, and 4) flowingsaid second solution into the reaction chamber; whereby said firstsolution and said second solution are mixed in the reaction chamber. 24.The method of claim 23 comprising the further step of closing said firstvalve.
 25. The method of claim 1, further comprising measuring change influorescence in the reaction chamber that results from combining thefirst and second solutions.